March 2006
Volume 47, Issue 3
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Cornea  |   March 2006
The Side Population Cells in the Rabbit Limbus Sensitively Increased in Response to the Central Cornea Wounding
Author Affiliations
  • Ki-Sook Park
    From the R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea; the
    Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Sciences, and the
  • Chae Ho Lim
    From the R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea; the
    Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Sciences, and the
  • Byung-Moo Min
    From the R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea; the
  • Jae Lim Lee
    From the R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea; the
  • Hee-Yong Chung
    Department of Microbiology, College of Medicine, Han Yang University, Seoul, Korea; and the
  • Choun-Ki Joo
    Department of Ophthalmology and Visual Science, College of Medicine, Catholic University of Korea, Seoul, Korea.
  • Chan-Woong Park
    From the R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea; the
  • Youngsook Son
    Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Sciences, and the
Investigative Ophthalmology & Visual Science March 2006, Vol.47, 892-900. doi:10.1167/iovs.05-1006
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      Ki-Sook Park, Chae Ho Lim, Byung-Moo Min, Jae Lim Lee, Hee-Yong Chung, Choun-Ki Joo, Chan-Woong Park, Youngsook Son; The Side Population Cells in the Rabbit Limbus Sensitively Increased in Response to the Central Cornea Wounding. Invest. Ophthalmol. Vis. Sci. 2006;47(3):892-900. doi: 10.1167/iovs.05-1006.

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

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Abstract

purpose. Side population (SP) cells are known to reside in the limbus as putative corneal epithelial stem cells. This study was performed to demonstrate the presence and the characteristics of SP cells in the rabbit limbal epithelium and explore their sensitivity in response to the central cornea wounding.

methods. To sort out the SP cells, freshly isolated rabbit limbal and central corneal epithelial cells were subjected to Hoechst 33342 dye efflux assay. For characterization of the sorted SP cells, RT-PCR analysis, semidry three-dimensional (3-D) cell culture, and transplantation in nude mice were performed. To explore wound sensitivity of the limbal SP cells, the rabbit central cornea was wounded by direct contact of a 6-mm paper disk soaked with 1 N NaOH, and changes in the population size of the SP cells and the colony-forming efficiency (CFE) was monitored on days 1, 2, and 5 after wounding.

results. The SP cells were present in the rabbit limbal epithelium with an incidence of 0.73% ± 0.14% (n = 8) and were smaller in cell size than the major population (MP) cells, quiescent in the cell cycle, and in the undifferentiated state. The SP cells were able to regenerate the cornealike structure with basal enrichment of p63-positive cells by in vitro 3-D culture and in vivo transplantation, all of which were best achieved by the whole population (WP) of cells comprising SP and MP cells. After central cornea wounding, this rare population of the limbal SP cells increased in size fivefold on day 1 and then decreased on day 2. The transient increase in the SP cells was subsequently followed by the propagation of an increase in CFE in the limbal MP cells on day 2 and then in the corneal MP cells on day 5. In the hematopoietic colony-forming assay, the limbal SP cells gave approximately eightfold higher CFU than the limbal MP cells.

conclusions. The SP cells identified in the rabbit limbus are an undifferentiated and noncycling rare epithelial cell population, which sensitively respond to the central cornea wounding by their transient increase in the population size.

Side population (SP) cells were first identified as a subpopulation of very primitive CD34-negative hematopoietic stem cells (HSCs) with long-term hematopoietic repopulation activity, which was characterized by their capacity to efflux Hoechst 33342 1 2 and was acquired by the expression of the ATP-binding cassette transporters such as Bcrp1/ABCG2. 3 4 5 Recently, the SP cells were also identified in muscle, 6 lung, 7 8 brain, 9 and liver 10 and this rare cell population was highly enriched for stem cell activity. Thus, the SP phenotype can be a useful criterion for the identification of tissue stem cells in a variety of species, because available molecular markers for the stem cell are still very limited. In contrast, several reports suggest that the SP phenotype may not be essential for stem cell function, because a qualitatively normal number of functional HSCs was detected in Bcrp1-deficient mice, 11 and the SP cells in the epidermis are a distinct subpopulation from the label-retaining cells (LRCs), satisfying general characteristics of keratinocyte stem cells. 12 13 Taken together, the SP phenotype cannot be a hallmark for the tissue stem cells unless other features of stem cells are concomitantly provided. 
In all self-renewing tissues, stem cells are postulated to exist in the slowly cycling state in a specialized area called as the niche and play a role as a reservoir that provides the progeny of rapidly dividing transit-amplifying (TA) cells, which are mainly responsible for the proliferation activities and population expansion of the tissue. 14 15 Therefore, stem cells are usually represented as LRCs and stem cells freshly isolated from the in vivo tissue under the normal resting state are quiescent in the cell cycle. 
Limbus localization of corneal epithelial stem cells has been suggested in the experimentally limbus-deficient rabbit due to the delayed cornea wound healing and recurrent erosion after repeated wounding. 16 Active participation of the limbus stem cells in acute cornea healing was shown by the report of Lehrer et al. 17 that by using double-labeling techniques, LRCs in the limbus are preferentially stimulated to be labeled with [3H]thymidine within 24 hours in response to the physical cornea wound that results in the depletion of differentiated cells. 17 Those previous reports strongly suggest that stem cells representative of LRCs may more sensitively respond to the wound challenge by entry into the cell cycle, to provide the extensive coverage needed for the corneal surface. 
The SP cells as putative corneal epithelial stem cells were reported in the human limbus, along with ABCG2 expression in the basal layer of the limbus, 18 19 in the primary cultured epithelial cells from the human limbus, 20 and in the rabbit limbus, with characteristic phorbol 12-myristate 13-acetate (PMA)-resistant colony-forming capacity. 21 However, the freshly isolated SP cells have not been fully explored because of the low incidence of such cells in the tissue and the lack of molecular markers for corneal stem cells in the rabbit. In this study, we demonstrated the presence of the SP cells in the freshly isolated rabbit limbus epithelial cells, and we characterized their differentiation status and their cell proliferation activity by RT-PCR analysis, with primers for cyclin B1, Ki67, connexin 43, and keratin 12, and their capacity to regenerate the stratified-cornea–like epithelium in vitro three dimensional (3-D) culture and in vivo transplantation. Finally, to explore the wound sensitivity of the limbus SP cells and its subsequent effect on the CFE capacity, the central cornea was wounded, and the changes in the SP cell pools and CFEs in the limbus and cornea were examined. 
Materials and Methods
Isolation of Rabbit Central Corneal and Limbal Epithelial Cells
New Zealand White rabbits weighing 2.5 to 3.0 kg were purchased from Samtako Biokorea (Seoul, Korea). The rabbits were anesthetized by an intramuscular administration of 10 mg/kg ketamine and 2.3 mg/kg xylazine and killed by asphyxiation with carbon dioxide. The cornea was surgically excised from the rabbit eye. All the animal experiments were approved by the ethics committee of the Korea Institute of Radiologic and Medical Sciences and were conducted in accordance with the ARVO Statement for the Use of Animal in Ophthalmic and Vision Research. After the endothelia were removed, the limbus was dissected from the central cornea by a criterion of an 8- to 9-mm diameter imaginary circle located from the center of eye, as defined previously. 22 23 24 25 Both the limbus and the central cornea tissues were washed with phosphate-buffered saline (PBS) containing 10 U/mL penicillin and 10 μg/mL streptomycin. The tissues were cut into four pieces and incubated with a 1:1 mixture of 2.4 U dispase II (Roche, Mannheim, Germany) in PBS and DMEM/F12 (3:1 mixture) containing 20% fetal bovine serum (FBS) for 16 hours at 4°C with gentle shaking. The detached epithelial sheets were incubated for an additional 20 minutes at 37°C with 0.5% trypsin and 0.54 mM EDTA (Invitrogen, Carlsbad, CA) with occasional vortexing. After twofold volume DMEM/F12 (3:1 mixture) containing 20% FBS was added, the epithelial cell suspension was centrifuged at 2200g and washed with DMEM/F12 (3:1 mixture) containing 2% FBS. 
Preparation of Rabbit Bone Marrow Cells
Femurs and tibias were isolated from the rabbit, and whole bone marrow cells were harvested by flushing femurs and tibias with ice-cold DMEM/F12 (3:1 mixture) containing 2% FBS by use of an 18-gauge needle and filtered through 70-μm nylon mesh. The filtered cells were harvested by centrifugation at 300g for 5 minutes, and the pellet was resuspended in 1 mL erythrocyte lysis buffer and incubated for 4 minutes on ice. After the cells were washed with DMEM/F12 (3:1 mixture) containing 2% FBS, they were used for the Hoechst 33342 efflux assay. 
Hoechst 33342 Efflux Assay
Freshly isolated bone marrow cells or epithelial cells from the limbus and central cornea were resuspended in DMEM/F12 (3:1 mixture) containing 2% FBS at a density of 1 × 106 cells/mL and incubated with 5 μg/mL Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) for 90 and 30 minutes at 37°C, respectively. To determine the verapamil-sensitive SP cells, we preincubated the bone marrow cells or the limbal and corneal epithelial cells with verapamil (5 or 50 μM, respectively) for 5 minutes before the addition of Hoechst 33342 dye. Immediately after they were stained, the cells were placed on ice and washed with ice-cold DMEM/F12 (3:1 mixture) containing 2% FBS. After an additional incubation for 30 minutes at 37°C to exclude Hoechst 33342, propidium iodide (PI; Sigma-Aldrich) was added at 2 μg/mL to gate out dead cells, and the cells were analyzed on a cell sorter (FACS Vantage; BD Biosciences, San Jose, CA) according to the method described by Goodell et al. 1 To prepare WP cells of the limbus or the central cornea, all the procedures of Hoechst 33342 staining, PI staining, and cell sorting were observed, to count the possible effect of UV exposure during the sorting procedure. Briefly, cells were excited at 350-nm, and emission was detected through a 450/20-nm narrow band-pass filter (Hoechst Blue) and 675-nm long-pass edge filter (Hoechst Red) with signal separated by a 610-nm short-pass dichroic mirror. Cells were then displayed in a Hoechst Blue versus Hoechst Red dot plot to visualize the SP cells. All the fluorescence data were collected in a linear mode and analyzed with flow cytometry acquisition and analysis software (CellQuest; BD Biosciences). To measure difference in cell sizes between the SP cells and the major population (MP) cells, a forward scatter (FSC) was used and analyzed (ModiFit Software; BD Biosciences). 
RT-PCR Analysis
Total RNA was extracted from corneal epithelial cells, the limbal SP cells, and the limbal MP cells (RNeasy Mini Kit; Qiagen, Hilden, Germany, and Absolutely RNA Nanoprep kit; Stratagene, La Jolla, CA), according to the manufacturers’ instructions. The cDNA was generated with reverse transcriptase (Superscript III; Invitrogen, Carlsbad, CA) and used for the gene amplification through a polymerase chain reaction (PCR; Perfect PreMix; Takara, Kyoto, Japan) with the gene-specific primers as summarized in Table 1 . The PCR products were resolved on 1.8% or 1.5% agarose gel with the 100-bp DNA marker (NEB, Beverly, MA). 
Central Cornea Wounding
After anesthesia, as described earlier, central cornea wounding was performed by applying a 6-mm diameter, 1 N NaOH-soaked filter paper onto the central cornea for 10 seconds and washed with excess PBS. Rabbits were killed on days 0, 1, 2, and 5 after wounding, and the rabbit central cornea containing a part of the peripheral cornea and the rabbit limbus were excised for cell isolation and histologic analysis. 
Semidry 3-D Cell Culture, Transplantation on the Back of Nude Mice, and Immunohistochemistry
A previous 3-D raft culture originally adopted for skin regeneration 30 was modified for the regeneration of stratified corneal epithelial equivalent. Briefly, Swiss 3T3 fibroblasts were embedded in the type I collagen gel matrix (Vitrogen; Angiotech BioMaterials, Palo Alto, CA) within a 12-mm insert (Millicell; Millipore, Billerica, MA) at a density of 2 × 105 cells/mL, and the cytometry-sorted cells were inoculated onto the stroma equivalent at densities of 500 cells (8.3 × 102 cells/cm2) or 10,000 cells per insert (1.67 × 104 cells/cm2) and cultured with E-medium (DMEM/F12 [3:1 mixture], containing 10% FBS, 5 μg/mL insulin, 5 μg/mL transferrin, 400 ng/mL hydrocortisone, 10−9 M cholera toxin, 10 U/mL penicillin, and 10 μg/mL streptomycin). After 3 weeks of submerged culture, the cells were exposed to a semidry condition by adjusting the medium level for an additional 2 weeks and were fixed with formalin. For the nude mouse transplantation, the 3-D cultures after the 3 weeks of submerged culture were implanted epithelial side up over the muscle fascia of nude mice (BALB/c Slc-nu/nu). 31 At 2 weeks after transplantation, the nude mice were euthanatized, and the tissue containing the corneal epithelial equivalents was excised. After fixation in formalin, paraffin-embedded sections (4 μm) were deparaffinized and hydrated, and the staining was performed as previously described. 32 The antibodies against p63 (Oncogene, Boston, MA), keratin 3/12 (Chemicon, Temecula, CA), and keratin 1/10 (Biomeda Corp., Foster City, CA) were applied according to the recommended titer supplied by the manufacturer. 
Epithelial CFE Assay
The cytometry-sorted cells were plated at a density of 100 cells per well on 6-well plates preseeded with Swiss 3T3 fibroblast feeder layers and cultured in the E-medium supplemented with 10 ng/mL EGF. After 12 days of culture, cells were fixed with 4% formaldehyde and stained with the mixture of 1% Nile blue and 1% rhodamine B. Colonies larger than 1-mm diameter (approximately >322 ± 74 cells per colony) were counted. The values of CFE were determined from the mean results of 10 independent experiments and calculated using the equation: colony forming efficiency (%) = (number of colonies/number of seeding cells) × 100. 
Hematopoietic CFU Assay
The sorted cells from bone marrow or the limbus were cultured in Iscove’s modified Dulbecco’s medium (IMDM) containing 1% methylcellulose, 30% FBS, 20 ng/mL human thrombopoietin, 50 ng/mL human stem cell factor, 50 ng/mL human Flt-3/Flk-2 ligand, 5 ng/mL mouse IL-3, 50 ng/mL mouse stem cell factor, and 5 ng/mL mouse granulocyte-macrophage colony-stimulating factor. Cells were plated in duplicate samples at a density of 3.5 × 104 cells per 35-mm culture dish. After 12 days, the number of colonies containing more than 50 cells was recorded. 
Results
Identification of SP Cells in the Adult Rabbit Limbal Epithelia with Small Cell Size
The SP cell-sorting method was first confirmed in freshly isolated rabbit bone marrow cells (Fig. 1A) . The SP cells, which were detected by verapamil-sensitive disappearance of a unique tail of a low Hoechst 33342 blue-red fluorescence, were successfully identified with the incidence of 1.67% ± 0.76% (n = 5) of bone marrow cells. From the freshly isolated limbal epithelial cells, the SP cells were identified based on their verapamil-sensitive disappearance, but no SP phenotype was detected in the corneal epithelial cells (Fig. 1B) . The incidence of the SP cells in the adult rabbit limbus was in the range of 0.54 to 0.92 with mean value of 0.73% ± 0.14% (n = 8). 
The stem cells, identified in a variety of tissues, are known to be smaller than the differentiated cells. The size distributions were compared by a forward scatter (FSC) parameter representing the cell size 9 33 (Fig. 1C) . The limbal SP cells were smallest in the cell size and sharply peaked (peak value of 200 FSC). In contrast, the limbal MP cells and the corneal epithelial cells were large and rather broadly distributed. The mean cell size increased in the order of the limbal SP, the limbal MP, and the central corneal epithelial cells. The limbal SP cells were smaller than the limbal MP cells and the corneal epithelial cells, similar to the previous reports. 21 33  
Behavior of the Limbal SP Cells in the Cell Cycle
The SP cells would be quiescent in the cell cycle immediately after their isolation from the tissue if the rabbit limbal SP cells were putative corneal epithelial stem cells. 15 Ki67 and cyclin B1 are known to be expressed in the actively cycling corneal epithelial cells, 34 35 and connexin 43 and keratin 12 are in the differentiated corneal epithelial cells. 19 36 37 RT-PCR analysis with the primers to Ki67, cyclin B1, connexin 43, and keratin 12 (Table 1)was performed (Fig. 2) . In contrast to the limbal MP cells and the central corneal epithelial cells, the limbal SP cells did not express either the cell cycle–associated genes or the differentiation-associated genes. Thus, the SP cells in the limbus are quiescent in the cell cycle and are undifferentiated. 
The Regeneration of Cornea- or Limbuslike Structures by the Limbal SP Cells, Limbal WP Cells, and Corneal WP Cells In Vitro and In Vivo
If the SP cells are putative corneal epithelial stem cells, they should reveal epithelial phenotype under the suitable condition. We tested the long-term repopulation capacity of the cytometry-sorted cells with semidry 3-D culture and nude mouse transplantation by seeding cells at a density as low as 8.3 × 102 cells/cm2 (Fig. 3A) . The limbal SP cells successfully regenerated the cornealike epithelium in vitro, with basal and suprabasal layers that were enriched with p63-positive cells, considered to be a marker of the corneal epithelial stem cell 38 and the corneal epithelial TA cells cultured in vitro 39 (Fig. 3B) . The limbal MP cells showed better corneal regeneration than the limbal SP cells only. In the limbus, WP cells comprised both SP and MP cells, were the most cornealike in morphology, had a basal clustered pattern of p63 expression, and had a suprabasal keratin 3/12 expression, similar to the in vivo limbus basal layer. The corneal WP cells at 8.3 × 102 cells/cm2 (500 cells/insert) also successfully replicated a cornealike structure with basal expression of p63. 
In the subcutaneous transplantation of the 3-D culture on the back of the nude mice (Figs. 3C 3D) , the limbal SP cells regenerated the stratified epithelia with basal expression of p63 and suprabasal expression of keratin 3/12 and without keratin 1/10 expression. In contrast, the limbal WP cells, even at a density of 8.3 × 102 cell/cm2 (500 cells/insert) regenerated the palisade of the Vogt (POV)-like structure similar to that of the limbus in vivo, where folded epithelium and intense p63 staining in the basal and suprabasal layers were clearly visible. The limbal MP cells only partially regenerated the POV-like structure. However, the corneal WP cells regenerated the cornealike tissue without the POV-like structure. The tissues regenerated in the nude mice did not express a skin-specified keratin 1/10. 
Wound-Sensitive Increase in Limbal SP Cells in the Limbus
It has been reported that the LRCs are predominantly located in the basal layer of the limbal epithelia and are preferentially stimulated to proliferate within 24 hours after central cornea wounding. 40 Alkali-burn central cornea wounding and its recovery were monitored as shown previously. 41 We investigated whether central cornea wounding can regulate the population size of the limbal SP cells (Fig. 4A) . The SP cell population in the limbus increased approximately fivefold on day 1 after wounding (3.68% ± 0.63%, n = 9, five independent experiments) over the population in the resting state, and its size was downregulated on day 2 (1.5% ± 1.48%, n = 9, five independent experiments; Fig. 4A ). In all experimental groups, the SP cell peak induction was shown on day 1, but the SP regression on day 2 was rather diverse between individuals. This transient peak induction of the SP cells in response to the central cornea wounding may also indicate that the SP cells are functionally working as putative stem cells of the corneal epithelia, similar to the previous report on the preferential cell cycle entry of the LRCs on central cornea wounding. 17  
Increase in CFE
The transient increase in the limbal SP cells after wounding may subsequently contribute to the overall increase in the TA cells of the limbus and then in the TA cells of the cornea. We compared the CFEs on different days after central cornea wounding (Fig. 4B) . The limbal SP cells from nonwounded eye or just after the wounding did not efficiently form the colony (Fig. 4B) . The CFE of the limbal MP cells (29.67% ± 3.93%) on day 0 was lower than that of the corneal WP cells (40.83% ± 4.07%), which is probably attributable to the peripheral cornea’s remaining after central cornea wounding. On day 1, the limbal SP cells generated colonies at 8% efficiency, compared with 0% CFE in the resting state. However, the CFEs of both limbal MP cells and corneal WP cells at the same time point decreased compared those on day 0. On day 2, the CFE of the limbal MP cells increased 1.67-fold compared with that on day 0, with induction sustained up to day 5 after the wounding. Then a large increase in the CFE of the corneal WP cells (approximately 80% CFE) followed on day 5. This surge of CFE in the cornea may have originated from the transient initial increase in the SP cells of the limbus on day 1 after wounding. 
Comparison of Epithelial Clonogenic Growth of Limbal SP and MP Cells in the HSC-CFU Assay Condition
The limbal SP cells freshly isolated from the rabbit limbus in the resting state did not efficiently generate the colony in the epithelial CFE assay condition (Fig. 4B) . To characterize further the culture preference of the limbal SP cells, the CFUs of the SP and MP cells from the bone marrow and the limbus were compared under conditions optimal for a hematopoietic CFU assay (Fig. 5A) . The bone marrow SP cells gave approximately 0.4% CFU, which was seven times higher than that of the bone marrow MP cells. Of note, the limbal SP cells generated much higher CFU (0.28%) than the limbal MP cells in the CFU assay conditions, approximately eight times higher. 
In contrast to the scattered hematopoietic morphology within the methylcellulose in the bone marrow SP cells, both the limbal SP cells and the limbal MP cells did not generate colonies within the methylcellulose but rather formed the colonies with an epithelial morphology on the surface of culture dishes. To rule out the possible contamination of keratocytes in the limbal SP population, we performed RT-PCR analysis with primer to lumican, expressed in keratocytes (Fig. 5C) . 42 43 No contamination of keratocytes in the initial limbal SP cell population was confirmed by lack of lumican expression. Again, colonies generated by the limbal SP cells were stained positively with pancytokeratin antibody (data not shown). 
Discussion
We identified the SP cells from the dispase II-dissociated epithelial sheet of the adult rabbit limbus that had been surgically dissected from the rest of the cornea. The limbal SP cells satisfied the SP criterion for the identification of bone marrow SP cells. Based on the characterization of the rabbit limbal SP cells in this study, the rabbit limbal SP cells are a rare and small epithelial subpopulation with the molecular nature of a noncycling and undifferentiated state, preferentially localized in the limbus. 
It has been suggested that corneal TA cells possess more expansion capacity than originally expected from other epithelial tissue, because the TA cells in the cornea can maintain the corneal coverage for more than 6 months in the limbus-deficient eye. 16 Also, corneal TA cells have a much higher population-doubling time than do other epithelial cells. 14 In this study, the MP cells derived either from the limbus or the cornea had much higher CFE than did the limbal SP cells in our epithelial clonogenic assay, which was preformed on a 14-day culture. Again, the CFEs of the resting limbal MP cells (30%), the resting corneal WP cells (40%), and the wound-stimulated corneal WP cells (80%) were considered to be much higher than expected from the possible number of corneal epithelial stem cells, even though the CFE assay adopted in this study is generally accepted for the evaluation of epithelial clonogenic potential. This result is also supported by the recent report that the rabbit limbal SP cells did not efficiently form colonies, 21 44 as well as our data that a higher CFU count in the HSC CFU assay was obtained in the limbal SP cells than in the limbal MP cells. Therefore, the epithelial CFE assay adopted in this study may not be optimal for determining the colony formation of corneal epithelial stem cells freshly isolated from the limbus tissue, which is quiescent in the cell cycle initially. With the HSC CFU assay as a basis, the optimal clonogenic assay for corneal epithelial stem cells may be developed in the future. 
According to a recent report, three isoforms of p63 are differentially expressed—ΔNp63α in the basal layer of the resting limbus and the β and γ isoforms of ΔNp63 in the active limbal as well as corneal epithelia and in vitro culture of limbal cells—suggesting that only an isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells. 45 In our 3-D cultures and nude mouse transplantation, limbal SP cell, limbal MP cell, limbal WP cells, and corneal WP cells, all of them showed p63 expressing cells in the suprabasal layer as well as the basal layer. However, all of the p63-expressing cells observed in this study may not be considered to be limbal stem cells, because the p63 antibody used study seems not to distinguish p63 isoforms. Therefore p63 expression per se does not show that those are epithelial stem cells in the regenerated tissues in vitro and in vivo. 
The primitive but inductive nature of the SP cells as tissue stem cells has been shown in the muscle-derived SP cells. 6 The muscle SP cells could display the muscle phenotype only when direct cellular contact with myoblasts is permitted. However, in the same condition, the bone marrow SP cells did not differentiate into myogenic lineage. Our data that the best regeneration of limbuslike epithelia such as the POV structure was shown in the in vivo transplantation of the limbal WP cells composed of the limbal SP cells and the limbal MP cells compared with those of the SP cells alone, the MP cells alone, or the corneal WP cells may suggest that the SP cells are very primitive but display the corneal epithelial stem cell phenotype only when the limbal MP cells are concomitantly provided along with the SP cells (Fig. 3) . However, the primitive and inductive nature of the limbal SP cells has to be shown based on the limbus regeneration capacity of the SP cells mixed with a different ratio of the MP cells in the future. 
Wound-sensitive cell cycle entry of LRCs within 24 hours after wounding was suggested as an indication that LRCs are corneal epithelial stem cells. 17 40 In this regard, a transient fivefold increase in the limbal SP cells in response to central cornea wounding (Fig. 4)provides the supporting evidence that the SP cells are putative corneal epithelial stem cells. A prompt fivefold induction of the SP cells may originate from the symmetric division of the limbal SP cells within a short period after wounding. If the wound-stimulated stem cell division is asymmetric, the stem cell pool may be maintained at a level similar to that of adult tissue in the resting state. It may also be considered that a transient increase in the population of the limbal SP cells and their rapid decrease results from insufficient downregulation of ABCG2 expression in the early TA cells, because of the rapid cell division and simultaneous entry into the TA cell fate of the wound-stimulated SP cells. However, this clarification was not possible in the rabbit model currently, because of the lack of ABCG2 gene information and other stem cell markers, but it may be elucidated in the future in this or another model. 
The wound-sensitive sequential increase in CFE from the limbal SP cells to limbal MP cells and then to corneal WP cells may suggest that a transient increase in the limbal SP cells is the early immediate response to the central cornea wound, which may be an essential first step to induction of the late response, such as an overall increase in CFE in the cornea, to meet the extensive requirement for corneal epithelial cells to compensate for tissue loss. In conclusion, the SP cells, preferentially located in the rabbit limbus, are a rare, undifferentiated, and noncycling subpopulation that sensitively responds to central cornea wounding by increasing their population size. 
 
Table 1.
 
Sequence of the Primers Used for Amplification in RT-PCR
Table 1.
 
Sequence of the Primers Used for Amplification in RT-PCR
Genes Primer Sequence Product Size (bp) Condition of RT-PCR
Keratin 12*
 Sense 5′-CACCGAGCGCCAGAACAT-3′ 542 34 Cycles of 30 seconds at 95°C, 30 seconds at 54°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-TCCAGGCCACCAGAAGAAAG
Connexin 43 26
 Sense 5′-CCCACGGAGAAAACCATCTT-3′ 538 34 Cycles of 30 seconds at 95°C, 30 seconds at 55°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-TCTCCAGGTCATCAGGCC-3′
Ki67 27
 Sense 5′-ACTTGCCTCCTAATACGCC-3′ 437 30 Cycles of 30 seconds at 94°C, 30 seconds at 45°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-TTACTACATCTGCCCATGA-3′
Cyclin B1 27
 Sense 5′-GGAGGAAGAGCAGTCAGTTA-3′ 275 35 Cycles of 30 seconds at 94°C, 30 seconds at 50°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-GTCACAAAAGCGAAGTCACC-3′
β-Actin 28
 Sense 5′-AAGATCTGGCACCACACCTT-3′ 125 34 Cycles of 30 seconds at 95°C, 30 seconds at 62°C, 45 seconds at 72°C and a final cycle of 7 minutes at 72°C
 Antisense 5′-CGAACATGATCTGGGTCATC-3′
Lumican 29
 Sense 5′-CTGCAGTGGCTCATTCAT-3′ 576 A Cycle of 5 minutes at 94°C and 23 cycles of 60 seconds at 94°C, 120 seconds at 55°C, 120 seconds at 72°C
 Antisense 5′-GACCTCCAGGTAATAGTT-3′
Figure 1.
 
Flow cytometric analysis of rabbit bone marrow cells, limbal epithelial cells, and central corneal epithelial cells. Rabbit bone marrow cells harvested from the femurs and tibias (A) and epithelial cells freshly isolated from the limbus and central cornea after the dispase-mediated removal of the stroma (B) were analyzed for Hoechst 33342 dye efflux by flow cytometry. The SP gate (black-boxed area) and the MP gate (red-boxed area) cells were marked. (C) Cell size distributions were compared by FSC parameters.
Figure 1.
 
Flow cytometric analysis of rabbit bone marrow cells, limbal epithelial cells, and central corneal epithelial cells. Rabbit bone marrow cells harvested from the femurs and tibias (A) and epithelial cells freshly isolated from the limbus and central cornea after the dispase-mediated removal of the stroma (B) were analyzed for Hoechst 33342 dye efflux by flow cytometry. The SP gate (black-boxed area) and the MP gate (red-boxed area) cells were marked. (C) Cell size distributions were compared by FSC parameters.
Figure 2.
 
RT-PCR analysis of the rabbit limbal SP cells, limbal MP cells, and central corneal epithelial cells. Proteins and expected sizes are shown. Marker: 100-bp DNA ladder. β-Actin (125 bp) was used as the internal control.
Figure 2.
 
RT-PCR analysis of the rabbit limbal SP cells, limbal MP cells, and central corneal epithelial cells. Proteins and expected sizes are shown. Marker: 100-bp DNA ladder. β-Actin (125 bp) was used as the internal control.
Figure 3.
 
Regeneration of cornea-or limbus-like structures in vitro and in vivo. (A) Experimental scheme for the semidry 3-D cell culture and for the nude mouse transplantation. (B) Hematoxylin and eosin (H&E) staining and immunohistochemical staining of the 3-D cell cultures with antibodies against p63 and keratin 3/12. (C) H&E staining of the nude mouse transplantation. The 3-D cultures, which were inoculated at a density of 500 cells/insert and submerged culture for 3 weeks, were transplanted for 2 weeks. Dotted line: The POV-like structure is dotted-lined. (D) Immunohistochemical staining with antibodies against p63, keratin 3/12, and keratin 1/10. Magnification: (B) ×200; (C) ×100; (D) ×400.
Figure 3.
 
Regeneration of cornea-or limbus-like structures in vitro and in vivo. (A) Experimental scheme for the semidry 3-D cell culture and for the nude mouse transplantation. (B) Hematoxylin and eosin (H&E) staining and immunohistochemical staining of the 3-D cell cultures with antibodies against p63 and keratin 3/12. (C) H&E staining of the nude mouse transplantation. The 3-D cultures, which were inoculated at a density of 500 cells/insert and submerged culture for 3 weeks, were transplanted for 2 weeks. Dotted line: The POV-like structure is dotted-lined. (D) Immunohistochemical staining with antibodies against p63, keratin 3/12, and keratin 1/10. Magnification: (B) ×200; (C) ×100; (D) ×400.
Figure 4.
 
The wound sensitivity of the limbal SP cell population and the effect on CFE of limbal SP cells, limbal MP cells, and corneal WP cells. The rabbit central cornea was injured by alkali burn. (A) Cytometric analysis of limbal epithelial cells isolated on a different day after the injury, with or without verapamil treatment. *Corneas of five rabbits were pooled to sort out SP cells through the Hoechst 33342 efflux assay. The results of five independent experiments are summarized. (B) Comparison of CFEs of the limbal SP cells, limbal MP cells, and corneal WP cells on different days after the injury. Right: representative colonies; left: data represent the mean CFEs.
Figure 4.
 
The wound sensitivity of the limbal SP cell population and the effect on CFE of limbal SP cells, limbal MP cells, and corneal WP cells. The rabbit central cornea was injured by alkali burn. (A) Cytometric analysis of limbal epithelial cells isolated on a different day after the injury, with or without verapamil treatment. *Corneas of five rabbits were pooled to sort out SP cells through the Hoechst 33342 efflux assay. The results of five independent experiments are summarized. (B) Comparison of CFEs of the limbal SP cells, limbal MP cells, and corneal WP cells on different days after the injury. Right: representative colonies; left: data represent the mean CFEs.
Figure 5.
 
CFU assay of the SP cells and the MP cells either from the bone marrow (BM) or the limbus (Lim). (A) Comparison of CFUs between the SP and MP cells either from the rabbit bone marrow or the rabbit limbus. (B) Morphologies of colonies. The bone marrow SP cells formed colonies within the methyl cellulose but the limbal SP cells formed colonies on the surface of the dish as an epithelial morphology. (C) RT-PCR analysis with primer to lumican. The expected size of lumican is 576 bp.
Figure 5.
 
CFU assay of the SP cells and the MP cells either from the bone marrow (BM) or the limbus (Lim). (A) Comparison of CFUs between the SP and MP cells either from the rabbit bone marrow or the rabbit limbus. (B) Morphologies of colonies. The bone marrow SP cells formed colonies within the methyl cellulose but the limbal SP cells formed colonies on the surface of the dish as an epithelial morphology. (C) RT-PCR analysis with primer to lumican. The expected size of lumican is 576 bp.
The authors thank Hee Jong Ahn and Jun-Sub Choi, PhD, at Catholic University for technical assistance of the flow cytometry and electron microscope analysis. 
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Figure 1.
 
Flow cytometric analysis of rabbit bone marrow cells, limbal epithelial cells, and central corneal epithelial cells. Rabbit bone marrow cells harvested from the femurs and tibias (A) and epithelial cells freshly isolated from the limbus and central cornea after the dispase-mediated removal of the stroma (B) were analyzed for Hoechst 33342 dye efflux by flow cytometry. The SP gate (black-boxed area) and the MP gate (red-boxed area) cells were marked. (C) Cell size distributions were compared by FSC parameters.
Figure 1.
 
Flow cytometric analysis of rabbit bone marrow cells, limbal epithelial cells, and central corneal epithelial cells. Rabbit bone marrow cells harvested from the femurs and tibias (A) and epithelial cells freshly isolated from the limbus and central cornea after the dispase-mediated removal of the stroma (B) were analyzed for Hoechst 33342 dye efflux by flow cytometry. The SP gate (black-boxed area) and the MP gate (red-boxed area) cells were marked. (C) Cell size distributions were compared by FSC parameters.
Figure 2.
 
RT-PCR analysis of the rabbit limbal SP cells, limbal MP cells, and central corneal epithelial cells. Proteins and expected sizes are shown. Marker: 100-bp DNA ladder. β-Actin (125 bp) was used as the internal control.
Figure 2.
 
RT-PCR analysis of the rabbit limbal SP cells, limbal MP cells, and central corneal epithelial cells. Proteins and expected sizes are shown. Marker: 100-bp DNA ladder. β-Actin (125 bp) was used as the internal control.
Figure 3.
 
Regeneration of cornea-or limbus-like structures in vitro and in vivo. (A) Experimental scheme for the semidry 3-D cell culture and for the nude mouse transplantation. (B) Hematoxylin and eosin (H&E) staining and immunohistochemical staining of the 3-D cell cultures with antibodies against p63 and keratin 3/12. (C) H&E staining of the nude mouse transplantation. The 3-D cultures, which were inoculated at a density of 500 cells/insert and submerged culture for 3 weeks, were transplanted for 2 weeks. Dotted line: The POV-like structure is dotted-lined. (D) Immunohistochemical staining with antibodies against p63, keratin 3/12, and keratin 1/10. Magnification: (B) ×200; (C) ×100; (D) ×400.
Figure 3.
 
Regeneration of cornea-or limbus-like structures in vitro and in vivo. (A) Experimental scheme for the semidry 3-D cell culture and for the nude mouse transplantation. (B) Hematoxylin and eosin (H&E) staining and immunohistochemical staining of the 3-D cell cultures with antibodies against p63 and keratin 3/12. (C) H&E staining of the nude mouse transplantation. The 3-D cultures, which were inoculated at a density of 500 cells/insert and submerged culture for 3 weeks, were transplanted for 2 weeks. Dotted line: The POV-like structure is dotted-lined. (D) Immunohistochemical staining with antibodies against p63, keratin 3/12, and keratin 1/10. Magnification: (B) ×200; (C) ×100; (D) ×400.
Figure 4.
 
The wound sensitivity of the limbal SP cell population and the effect on CFE of limbal SP cells, limbal MP cells, and corneal WP cells. The rabbit central cornea was injured by alkali burn. (A) Cytometric analysis of limbal epithelial cells isolated on a different day after the injury, with or without verapamil treatment. *Corneas of five rabbits were pooled to sort out SP cells through the Hoechst 33342 efflux assay. The results of five independent experiments are summarized. (B) Comparison of CFEs of the limbal SP cells, limbal MP cells, and corneal WP cells on different days after the injury. Right: representative colonies; left: data represent the mean CFEs.
Figure 4.
 
The wound sensitivity of the limbal SP cell population and the effect on CFE of limbal SP cells, limbal MP cells, and corneal WP cells. The rabbit central cornea was injured by alkali burn. (A) Cytometric analysis of limbal epithelial cells isolated on a different day after the injury, with or without verapamil treatment. *Corneas of five rabbits were pooled to sort out SP cells through the Hoechst 33342 efflux assay. The results of five independent experiments are summarized. (B) Comparison of CFEs of the limbal SP cells, limbal MP cells, and corneal WP cells on different days after the injury. Right: representative colonies; left: data represent the mean CFEs.
Figure 5.
 
CFU assay of the SP cells and the MP cells either from the bone marrow (BM) or the limbus (Lim). (A) Comparison of CFUs between the SP and MP cells either from the rabbit bone marrow or the rabbit limbus. (B) Morphologies of colonies. The bone marrow SP cells formed colonies within the methyl cellulose but the limbal SP cells formed colonies on the surface of the dish as an epithelial morphology. (C) RT-PCR analysis with primer to lumican. The expected size of lumican is 576 bp.
Figure 5.
 
CFU assay of the SP cells and the MP cells either from the bone marrow (BM) or the limbus (Lim). (A) Comparison of CFUs between the SP and MP cells either from the rabbit bone marrow or the rabbit limbus. (B) Morphologies of colonies. The bone marrow SP cells formed colonies within the methyl cellulose but the limbal SP cells formed colonies on the surface of the dish as an epithelial morphology. (C) RT-PCR analysis with primer to lumican. The expected size of lumican is 576 bp.
Table 1.
 
Sequence of the Primers Used for Amplification in RT-PCR
Table 1.
 
Sequence of the Primers Used for Amplification in RT-PCR
Genes Primer Sequence Product Size (bp) Condition of RT-PCR
Keratin 12*
 Sense 5′-CACCGAGCGCCAGAACAT-3′ 542 34 Cycles of 30 seconds at 95°C, 30 seconds at 54°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-TCCAGGCCACCAGAAGAAAG
Connexin 43 26
 Sense 5′-CCCACGGAGAAAACCATCTT-3′ 538 34 Cycles of 30 seconds at 95°C, 30 seconds at 55°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-TCTCCAGGTCATCAGGCC-3′
Ki67 27
 Sense 5′-ACTTGCCTCCTAATACGCC-3′ 437 30 Cycles of 30 seconds at 94°C, 30 seconds at 45°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-TTACTACATCTGCCCATGA-3′
Cyclin B1 27
 Sense 5′-GGAGGAAGAGCAGTCAGTTA-3′ 275 35 Cycles of 30 seconds at 94°C, 30 seconds at 50°C, 45 seconds at 72°C, and a final cycle of 10 minutes at 72°C
 Antisense 5′-GTCACAAAAGCGAAGTCACC-3′
β-Actin 28
 Sense 5′-AAGATCTGGCACCACACCTT-3′ 125 34 Cycles of 30 seconds at 95°C, 30 seconds at 62°C, 45 seconds at 72°C and a final cycle of 7 minutes at 72°C
 Antisense 5′-CGAACATGATCTGGGTCATC-3′
Lumican 29
 Sense 5′-CTGCAGTGGCTCATTCAT-3′ 576 A Cycle of 5 minutes at 94°C and 23 cycles of 60 seconds at 94°C, 120 seconds at 55°C, 120 seconds at 72°C
 Antisense 5′-GACCTCCAGGTAATAGTT-3′
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