KR100804009B1 - Methods for Culturing Limbal Side Population Cells - Google Patents

Abstract

The present invention comprises the steps of (a) separating the epithelial sheet by treatment with dispase on the separated limbus; (b) treating the isolated epithelial sheet with a trypsin and EDTA mixture to obtain a cell suspension comprising corneal lead epithelial cells; And (c) sorting the corneal lead SP cells from the cell suspension containing the corneal lead epithelial cells, the method for separating corneal lead SP (side population) cells, a composition for separating corneal lead SP cells, and cornea. A method for regenerating epithelium and regenerated artificial corneal epithelium. According to the present invention, corneal smoked SP cells can be separated with high efficiency, and corneal smoked SP cells can be successfully used to reconstruct corneal epithelium.

Corneal smoke, SP cells, Corneal epithelium, 3D culture, Dispase, Trypsin, EDTA

Description

Method for Culturing Limbal Side Population Cells

The present invention relates to a method for separating corneal lead SP cells, a composition for separating corneal lead SP cells, a method for regenerating corneal epithelium, and a regenerated artificial corneal epithelium.

Side population (SP) cells were first identified as early CD34-negative hematopoietic stem cells with long-term hematopoietic repopulation activity, characterized by the ability to release Hoechst 33342 [1,2], Bcrp1 / By expression of an ATP-binding cassette transporter such as ABCG2. Recently, tissue SP cells were identified in muscle [6], lung [7,8], brain [9] and liver [10]. The rare cell population is enriched for stem cell activity and is known to successfully repair tissue damage regardless of origin when injected into damaged tissue or irradiated bone marrow. For example, muscle bone marrow was reconstructed using muscle SP cells [11]. In contrast, bone marrow SP cells were used to treat myocardial damage and activity to partially restore dystrophin expression in the injured muscle of mdx mice. Activity was shown [11,12].

In contrast to the in vivo data, cultured muscle SP cells showed mostly HSC properties instead of muscle phenotype unless co-cultured with tissue specific cells such as myoblasts [6]. However, under the same conditions, myeloid SP cells did not acquire muscle properties. This fact suggests that tissue SP cells are cells of a different nature from bone marrow SP, although they have features in common with bone marrow SP, and are early stem cells that are instructed to acquire tissue properties by damaged tissue environments or neighboring cells under coculture conditions. Indicates that

The SP phenotype is useful as a criterion for the identification of tissue stem cells in various species because the molecular markers available for stem cells are very limited. However, normal numbers of functional HSCs were detected in Bcrp1-deficient mice [13], indicating that the SP phenotype is not essential for stem cell function. Unlike SP cells in other tissues, SP cells in the epidermal layer are clearly different subpopulations with marker-bearing cells that meet the general characteristics of keratinous stem cells, such as high expression of β1 integrins [14]. The basic level of ABCG2 expression, ATP-binding cassette transporter, was low in the basal cell layer of the skin where epithelial stem cells were supposed to be located, but high in glands secretory epithelium [15]. Taken together, it can be seen that the SP phenotype cannot be a reference for tissue stem cells unless given other characteristics of stem cells.

In all self-renewing tissues, stem cells are present at a specific site called niche and are the progeny of rapidly dividing transit-amplifying (TA) cells (which are involved in the major proliferative activity and population expansion of tissues). It serves as a reservoir for supplying [16,17]. Stem cells are relatively undifferentiated and are also arrested in the cell cycle under resting conditions [18]. However, upon wounding, these resting stem cells enter the cell cycle within 24 hours, observed by DNA double-labeling with ³ H-thymidine in the BrdU label-bearing cells (LRCs) of rat corneas. [19]. Thus, wound responsiveness may be a functional criterion for the identification of stem cells, particularly corneal epithelial stem cells.

SP cells as putative corneal epithelial stem cells were first identified in human cornea with ABCG2 expression in the basal layer of corneal margin [20, 21]. However, not enough studies have been done to support the putative corneal epithelial stem cells.

Throughout this specification, a number of citations are referenced and citations are indicated. The disclosures of the cited documents are hereby incorporated by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors conducted an in-depth study to develop a method for efficiently separating corneal smoke SP (side population) cells as a material for regenerating corneal epithelium. The present invention has been accomplished by reproducing corneal epithelium successfully using the isolated corneal lead SP cells.

Accordingly, it is an object of the present invention to provide an efficient method for separating corneal lead SP cells.

Another object of the present invention to provide a composition for separating corneal lead SP cells.

Still another object of the present invention is to provide a method for regenerating corneal epithelium using corneal lead SP cells.

Another object of the present invention is to provide a regenerated artificial corneal epithelium.

Another object of the present invention to provide a method for culturing corneal lead SP cells.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for separating corneal edema SP (side population) cells comprising the following steps: (a) Epithelium by treatment with dispase on the isolated limbus; Separating the sheet; (b) treating the isolated epithelial sheet with a trypsin and EDTA mixture to obtain a cell suspension comprising corneal lead epithelial cells; And (c) sorting the corneal lead SP cells from the cell suspension comprising the corneal lead epithelial cells.

According to another aspect of the present invention, the present invention provides a composition for isolation of corneal marginal SP (side population) cells comprising trypsin, EDTA and dispase.

According to another aspect of the present invention, the present invention provides a method for regeneration of corneal epithelium using corneal lead SP cells, comprising the steps of: (a) treating the separated cornea with dispase; Separating the epithelial sheet; (b) treating the isolated epithelial sheet with a trypsin and EDTA mixture to obtain a cell suspension comprising corneal lead epithelial cells; (c) sorting corneal lead SP cells from a cell suspension comprising the corneal lead epithelial cells; (d) seeding a cell suspension comprising said corneal lead SP cells on a feeder cell layer circulated in a type I collagen gel matrix; And (e) culturing the cell suspension comprising corneal margin SP cells of step (d) in a semi-dry state to obtain a multilayer corneal epithelium.

According to another aspect of the present invention, the present invention provides artificial corneal epithelium regenerated by the method of the present invention described above.

The present inventors conducted an in-depth study to develop a method for efficiently separating corneal smoke SP (side population) cells as a material for regenerating corneal epithelium. It was confirmed that it shows a very high efficiency in the separation of cells. And corneal epithelium was successfully regenerated using the isolated corneal lead SP cells.

As used herein, the term “limbus side population cell” refers to two-dimensional fluorescence intensity after fluorescence staining of corneal lead epithelial cells with a Hoechst 33342 staining agent. Coordinate the fluorescence intensity of 450 nm means that cell populations are observed in the lower left of the coordinates (ie when the fluorescence intensity at 450 nm and 675 nm are both low), which means Goodell, MA et. al., J. Exp . Med . , 183: 1797-1806 (1996)). These corneal limbal SP cells are greatly reduced by verapamil treatment, undifferentiated and stationary in cell cycle, and do not express Ki67, cyclin B1, connexin 43 and keratin 12.

The invention is described in more detail in each step:

I. Preparation of Cell Suspensions Containing Corneal Smokey Epithelial Cells

Corneal marginal epithelial cells are isolated from the corneal margin located between the cornea and the conjunctiva. This corneal smoked epithelial cell contains corneal smoked SP cells. Referring to this step according to a specific embodiment of the present invention are as follows:

First, the corneal smoke is cut into small pieces, and then dispase treatment to separate the corneal smoked epithelial sheet. Dispase used in this process is Bacillus-derived (eg, Bacillus polymyxa ) metalloprotease. Dispase is used for various purposes in tissue engineering. In the present invention, it is used to dissociate corneal marginal epithelial sheet tissue from corneal margins. Preferably, the dispase is dispase II. De Spa to azepin process is preferably Mg ^{2 +} and Ca ^{2 +} - is carried out in a buffer member. As the buffer, various buffers known in the art may be used. Preferably, the buffers exhibit buffering power in the range of pH 6-8. Most preferably, phosphate buffered saline (PBS). The amount of dispase to be treated is 0.5-4 units, preferably 1.0-3.0 units, more preferably 2.0-3.0 units, most preferably about 2.5 units. The dispase treatment process is generally carried out at low temperatures, preferably at 3-15 ° C., more preferably at 3-10 ° C. and most preferably at 4-6 ° C. The dispase reaction time is relatively long, 10-24 hours, preferably 10-20 hours, more preferably 12-18 hours, most preferably 14-18 hours.

Subsequently, the isolated corneal smoked epithelial sheet is treated with a mixture of trypsin and EDTA to obtain corneal smoked epithelial cells into single cells. The trypsin used is 0.1-1.5% trypsin, preferably 0.1-1.0% trypsin, more preferably 0.2-0.8% trypsin, most preferably 0.4-0.6% trypsin and the concentration of EDTA used is 0.1-1.5 mM , Preferably 0.1-1.0 mM, more preferably 0.2-0.8 mM, most preferably 0.5-0.6 mM. The reaction temperature is preferably carried out at the general enzyme reaction temperature (30-40 ℃), the reaction time is 10-60 minutes, preferably 20-50 minutes, more preferably 20-40 minutes, most preferably 20 -25 minutes. If the reaction time is 10 minutes or less, the trypsin / EDTA treatment effect is hardly exhibited. If it exceeds 60 minutes, the cell viability is adversely affected.

In the method of the present invention with dispase treatment followed by trypsin / EDTA treatment, dispase acts on the interface between the basal surface of the epithelial cell layer and the basement membrane, and trypsin / EDTA is the basal epithelium. Epithelial cells can be obtained with high efficiency because they act on the intercellular connections between cells.

II. Corneal smoke Sorting of SP Cells

The corneal lead SP cells are then sorted from a cell suspension comprising corneal lead epithelial cells.

Sorting of corneal lead SP cells was performed using the Goodell method known in the art (Goodell, MA et al., J. Exp . Med . , 183: 1797-1806 (1996)), i.e. after staining FACS (fluorescence- activated cell sorting). More specifically, 5 μg / ml Hoechst 33342 is treated in a corneal soft epithelial suspension of 1 × 10 ⁶ cells / ml and reacted at 37 ° C. for 90 or 30 minutes. Propidium iodide (PI) is then added at 2 μg / ml, followed by sorting SP cells using FACS cell sorter. For sorting of SP cells, 450/20 nm narrowband pass filter (Hoechst Blue) and 675 nm long pass mouse filter (Hoechst Red) with signals excited at 350 nm and separated by a 610 nm short pass dichroic mirror To detect the emission signal. The cells were then hoechst blue vs. SP cells are identified by Hoechst Red dot plot. SP cells are located in the bottom left (ie, when the fluorescence intensity at both 450 nm and 675 nm is low) in the dot plot (see FIGS. 1A-1B).

These sorted SP cells can be reconfirmed by examining the verapamil sensitivity in pest staining, Ki67, cyclin B1, Connexin 43 and keratin 12 not expressing.

The method of the present invention makes it possible to separate corneal lead SP cells with very high efficiency, which is not achieved by any conventional technique.

III. Corneal smoke Reproduction of epithelial cells using the SP (regeneration)

When regenerating the three-dimensional (3-D) corneal epithelium using a cell suspension containing corneal lead SP cells obtained by the above procedure, the corneal lead SP cells are fed into a type I collagen gel matrix. Seeding on stromal cells, dermal fibroblasts, 3T3 cells or STO fibroblasts] layer. In addition, it is preferable for 3-D culture that the type I collagen gel matrix is in a millicell having a three-dimensional structure.

The medium used for 3-D culture of corneal lead SP cells may be any conventionally used in the art for animal cell culture. According to the most preferred embodiment of the present invention, a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is used. This medium includes accessory ingredients commonly used in the art for the proliferation of animal cells, such as serum (eg fetal calf serum), insulin, transferrin, hydrocortisone, cholera toxin, penicillin and / or streptomycin. .

The culture conditions of seeded corneal lead SP cells can be largely selected according to air exposure: air-dry conditions, semi-dry conditions and submerged conditions. .

Air-drying conditions herein refer to a condition where corneal smoked SP cell layers seeded on feeder cell layers embedded in a type I collagen gel matrix are completely exposed to air (see panel A of FIG. 3A).

Semi-drying conditions as used herein refer to a condition in which a corneal margin SP cell layer seeded on a feeder cell layer cultivated in a type I collagen gel matrix is covered with a culture medium of the lowest possible height (see panel B of FIG. 3A). In this condition, the kerchief solution is covered as thinly as possible just above the corneal lead SP cell layer, and the factors affecting the corneal lead SP cell layer act through the culture medium and the air.

Submerging conditions herein refer to a condition where the corneal lead SP cell layer seeded on the feeder cell layer circulated in the type I collagen gel matrix is covered with the culture medium of the highest possible height (see Panel C of FIG. 3A). In this condition, factors affecting the corneal lead SP cell layer act through the culture medium.

Cultivation of seeded corneal lead SP cells is largely carried out in two stages. The first incubation step is carried out under submerging conditions, which are common culture conditions. The duration of the first incubation is 1-30 days, preferably 5-30 days, more preferably 10-25 days, most preferably 18-23 days. Medium conditions are the same as described above. The second incubation step is carried out in semi-dry conditions. The duration of the second incubation is 1-20 days, preferably 5-20 days, more preferably 10-18 days, most preferably 12-16 days.

Seed corneal stem cells are cultured on the feeder cell layer. The feeder cell layer may exhibit inert mitotically active and may exhibit mitotically active. Preferably, the feeder cell layer is mitotically active. The method of making mitotic inactivation may be by gamma-ray irradiation or mitomycin C treatment, preferably the mitotic inactive feeder cell layer is by mitomycin C-treatment. The kind of feeder cells that can be used may be any cell known in the art, preferably fibroblasts (eg dermal fibroblasts, 3T3 fibroblasts or STO fibroblasts) and stromal cells, most preferably Fibroblasts.

According to a preferred embodiment of the present invention, the cell suspension comprising the corneal lead SP cells of step (d) further comprises corneal lead (MP) cells. When seeding the cells seeded on the feeder cell layer in combination with corneal lead MP cells, the seeded cells are more efficiently regenerated than when the seeded cells are composed of corneal lead SP cells alone. As used herein, the term "corneal lead MP cell" refers to the remaining cell population except for SP cells in the sorting process of corneal lead SP cells from the aforementioned corneal lead epithelial cells.

According to the corneal epithelial regeneration method of the present invention, it is possible to artificially reconstruct the corneal epithelium efficiently.

According to another aspect of the present invention, the present invention provides a method for treating corneal lead SP cells by (i) stem cell factor, (ii) granulocyte-macrophage colony-stimulating factor (GM-CSF), and (iii) IL-3. It provides a method for culturing corneal smoke SP cells comprising culturing in a medium containing (interleukin-3).

In order to establish an efficient method for culturing corneal lead SP cells, the present inventors examined the culture preference, and as a result, when cultured under hematopoietic colony forming conditions, it was cultured more efficiently, especially than corneal lead (major population) cells. I found it possible.

A feature of the culture method of the present invention is that the stem cell factors, GM-CSF and IL-3, are used as cytokine components of the medium. The cytokine is a component used when culturing hematopoietic stem cells to form colonies. The amount of stem cell factor is 10-300 ng / ml, preferably 10-200 ng / ml, more preferably 10-100 ng / ml, most preferably about 30-60 ng / ml, the GM- The amount of CSF is 1-30 ng / ml, preferably 1-20 ng / ml, more preferably 1-10 ng / ml, most preferably about 3-6 ng / ml, and the amount of IL-3 1-30 ng / ml, preferably 1-20 ng / ml, more preferably 1-10 ng / ml, most preferably about 3-6 ng / ml.

In addition to the cytokine component, the medium used in the present invention includes a basic medium commonly used for animal cell culture in the art, such as DMEM, Ham's F12 medium, IMDM (Iscove's Modified Dulbecco's Media) or RPMI. According to a preferred embodiment of the invention, the medium of the invention additionally comprises serum (eg fetal calf serum). In addition, the medium of the present invention, in addition to the cytokine, such as IL-4, IL-13, IL-7, TGF-β ₁ (Transforming growth factor-beta 1) and / or TNF-α (Tumor necrosis factor-alpha) It may further comprise a cytokine.

Conditions employed in the culturing step of the present invention is generally the conditions used for the culture of animal cells.

According to the method of the present invention, corneal lead SP cells can be cultured efficiently, especially more predominantly than corneal lead MP cells.

As described in detail above, the present invention provides an efficient method for separating corneal lead SP cells and compositions for separating corneal lead SP cells. The present invention also provides a corneal epithelial regeneration method using corneal lead SP cells and artificial corneal epithelium. According to the present invention, corneal smoked SP cells can be separated with high efficiency, and corneal smoked SP cells can be successfully used to reconstruct corneal epithelium.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental Materials and Methods

Rabbit cornea and Corneal smoke  Isolation of Epithelial Cells

2.5-3.0 kg of New Zealand white rabbits were purchased from Samtako Biokorea (Seoul, Korea). Rabbits were anesthetized by intramuscular injection of 10 mg / kg ketamine and 2.3 mg / kg xylazine, followed by sacrifice in a carbon dioxide asphyxiation mode. The cornea was surgically extracted from rabbit eyes. All animal experiments were approved by the Ethics Committee of Korea Atomic Energy Research Institute and conducted in accordance with the ARVO Declaration on Animal Use by Ophthalmic and Vision Research.

After removal of the endothelium, corneal margins were separated from the central cornea. Corneal smoke and central corneal tissue were washed with PBS containing 10 units / ml penicillin and 10 μg / ml streptomycin. The tissue is cut into small pieces (6 pieces) and DMEM / F12 (3: 1 mixture) containing 2.5 U dispase II (Roche; Mannheim, Germany) and 20% fetal bovine serum (FBS) in PBS. The mixture was reacted gently with a 1: 1 mixture of at 16 ° C. for 16 hours. The isolated epithelial sheet was reacted with 0.5% trypsin-0.54 mM EDTA (Invitrogen; Carlsbad, Calif.) For an additional 20 minutes, often vortexing at 37 ° C. 2 volumes of DMEM / F12 (3: 1 mixture) containing 20% FBS were added, then the epithelial cell suspension was centrifuged at 2200 xg and washed with DMEM / F12 (3: 1 mixture) containing 2% FBS. .

Preparation of Rabbit Bone Marrow Cells

Isolate the femur and tibia from the rabbit, and then collect the whole bone marrow cells by flushing the femur and tibia using an 18-gauge needle with ice-cold DMEM / F12 (3: 1 mixture) containing 2% FBS, Filtered with 80 μm nylon mesh. The filtered cells were collected by centrifugation at 300 x g for 5 minutes, then the pellet was resuspended in 1 ml erythrocyte lysate buffer and placed on ice. After washing with DMEM / F12 (3: 1 mixture) containing 2% FBS, rabbit myeloid cells were used for Hoechst 33342 release assay.

Hoechst  33342 emission analysis

Freshly isolated bone marrow cells or epithelial cells isolated from corneal margin and central cornea were resuspended in DMEM / F12 (3: 1 mixture) containing 2% FBS at a density of 1 x 10 ⁶ cells / ml, followed by 5 μg / Reaction with ml Hoechst 33342 (Sigma; St. Louis, Mo.) at 37 ° C. for 90 or 30 minutes. To identify verapamil-sensitive SP cells, bone marrow cells or corneal margins and corneal epithelial cells were pre-reacted with verapamil (5 μM or 50 μM) for 5 minutes before adding Hoechst 33342 staining reagent. Immediately after staining the cells were placed on ice and washed with ice-cold DMEM / F12 (3: 1 mixture) containing 2% FBS. To exclude Hoechst 33342, additional reaction was carried out at 37 ° C. for 30 minutes, followed by addition of propidium iodide (PI) (Sigma; St Louis, MO) at 2 μg / ml, followed by Goodell et al . According to the method of [1] was analyzed using FACS Vantage cell sorter (BD Biosciences; San Jose, CA). In order to prepare WP cells of cornea margin or central cornea, Hoechst 33342 staining and PI staining were performed in the same manner as the above method, and FACS sorting was omitted. Briefly, the emission signals were detected with a 450/20 nm narrowband pass filter (Hoechst Blue) and a 675 nm long pass egest filter (Hoechst Red) with signals excited at 350 nm and separated by a 610 nm short pass dichroic mirror. It was. Cells were Hoechst Blue vs. SP cells were identified by the Hoechst Red dot plot. All fluorescence data were digitized in linear mode and analyzed with CellQuest flow cytometry acquisition / analysis software (BD Biosciences; San Jose, Calif.). To determine the difference in size of SP cells and MP cells, omnidirectional scatter (FSC) was used and analyzed by ModiFit software (BD Biosciences; San Jose, Calif.).

RT - PCR  analysis

Total RNA was isolated from corneal epithelial cells, corneal lead SP cells and corneal lead MP cells using the RNeasy Mini kit (Qiagen; Hilden, Germany) and the Absolutely RNA Nanoprep kit (Stratagene; La Jolla, Calif.). cDNA was generated with Superscript III reverse transcriptase (Invitrogen; Carlsbad, Calif.) and then PCR amplified using gene-specific primers and Perfect PreMix (Takara; Kyoto, Japan). Sense and antisense primers specific for rabbit Ki67, rabbit keratin 12, rabbit connexin 43 and rabbit β-actin are summarized in Table 1.

gene Primer Type Primer sequence Product size (bp) Keratin 12 ^* sense 5'-CACCGAGCGCCAGAACAT-3 ' 542 Antisense 5’-TCCAGGCCACCAGAAGAAAG-3 ’ Connexin 43 [22] sense 5'-CCCACGGAGAAAACCATCTT-3 ' 538 Antisense 5’-TCTCCAGGTCATCAGGCC-3 ’ Ki [23] sense 5'-ACTTGCCTCCTAATACGCC-3 ' 437 Antisense 5’-TTACTACATCTGCCCATGA-3 ’ Cyclin B1 [23] sense 5'-GGAGGAAGAGCAGTCAGTTA-3 ' 275 Antisense 5’-GTCACAAAAGCGAAGTCACC-3 ’ β-actin [24] sense 5'-AAGATCTGGCACCACACCTT-3 ' 125 Antisense 5’-CGAACATGATCTGGGTCATC-3 ’ ^* GenBank Accession Number, X77665

Conditions for cyclin B1 were as follows: 35 cycles in total, 94 ° C. 30 seconds, 50 ° C. 30 seconds, 72 ° C. 45 seconds and 72 ° C. 10 minutes in the final cycle. PCR conditions for Ki67 are as follows: 30 cycles in total, 94 ° C. 30 seconds, 45 ° C. 30 seconds, 72 ° C. 45 seconds and 72 ° C. 10 minutes in the final cycle. PCR conditions for keratin 12 were as follows: 34 cycles in total, 95 ° C. 30 sec, 54 ° C. 30 sec, 72 ° C. 45 sec and 72 ° C. 10 min in the final cycle. PCR conditions for Connexin 43 are as follows: 34 cycles in total, 95 ° C. 30 seconds, 55 ° C. 30 seconds, 72 ° C. 45 seconds and 72 ° C. 10 minutes in the final cycle. PCR conditions for β-actin are as follows: total 34 cycles, 95 ° C. 30 sec, 62 ° C. 30 sec, 72 ° C. 45 sec and 72 ° C. 7 min in the final cycle. PCR products were resolved with 100 bp DNA markers (NEB; Beverly, MA) on 1.8% or 1.5% agarose gels.

In the central cornea  Wounding ( wounding )

Rabbits were anesthetized by intramuscular administration of 10 mg / kg ketamine and 2.3 mg / kg xylazine. A 6 mm diameter filter paper soaked with 1 N NaOH was placed on the central cornea for 10 seconds to remove the central corneal epithelium and then washed with PBS. After the wound cycle, chloramphenicol was administered once daily until sacrifice. Rabbits were sacrificed at 0, 1, 2, and 5 days after wounding, and a portion of the peripheral cornea and the central cornea including the corneal margin were separated and used for cell separation and histological analysis.

Semi-Dry 3-D Cell Culture

The 3-D raft culture method [25], which was originally used for skin regeneration, was modified to regenerate a multilayer corneal epithelial equivalent. As a corneal parenchymal equivalent, feeder cells were seeded in a type I collagen gel matrix at a density of 2 × 10 ⁵ cells / ml in 12 mm millicells (Millipore, Billerica, Mass., USA). 200000 cells per ml of collagen matrix gel when using Swiss 3T3 fibroblasts (ATCC) as feeder cells, 200000 cells per ml of collagen matrix gel when using dermal fibroblasts, and collagen when using corneal stroma. 200000 cells were used per ml of matrix gel.

Corneal lead SP cells, corneal lead MP cells, corneal lead WP cells and corneal WP cells were plated on the corneal parenchyma homogenates at densities of 500 and 10,000 cells / millicell, in this case 10% FBS, 5 μg / DMEM and Ham's F12 medium (3: 1 mixture) containing ml insulin, 5 μg / ml transferrin, 400 ng / ml hydrocortisone, ^10-9 M cholera toxin, 10 units / ml penicillin and 10 μg / ml streptomycin Was used. After three weeks of submerge incubation, the media height was adjusted to expose the culture to semi-dry conditions for two weeks. Cultures were fixed for histology and immunohistochemical staining.

Transplantation on the back of nude mice

Corneal epithelial equivalents cultured under submerging conditions for 3 weeks were implanted subcutaneously into the dorsal skin of nude mice (BALB / cSlc-nu / nu). Under anesthesia, an incision was made in the dorsal skin of the nude Mau and the corneal epithelial equivalent was implanted on top of the fascia [26]. Two weeks after transplantation, nude mice were euthanized with carbon dioxide and tissues containing corneal epithelial equivalents were dissected and used for immunohistochemical analysis.

Immunohistochemical Staining

Paraffin was removed from the paraffinic cuts (4 μm) of the 3-D culture and the transplanted tissue and then hydrated. Staining was carried out according to a conventionally known method [27]. An antibody against p63 (Oncogene; Boston, MA) was used as a titer recommended by the manufacturer.

Hematopoiesis Colony -Forming units ( CFU ) analysis

Older bone marrow SP cells, bone marrow MP cells, corneal lead SP cells or corneal lead MP cells were isolated from 1% methylcellulose, 30% FBS, 5 ng / ml mouse IL-3, 50 ng / ml mouse stem cells. Factor, 5 ng / ml mice were cultured in Iscove's Modified Dulbecco's Media (IMDM) containing a granulocyte-macrophage colony-stimulating factor (GM-CSF). Two sets of cells were plated at a density of 3.5 × 10 ⁴ cells per 35 mm culture dish. After 12 days, the number of colonies containing at least 50 cells was counted.

epithelium Colony  Forming efficiency ( CFE ) analysis

After separation, old corneal lead SP cells, corneal lead MP cells and corneal WP cells were plated at a density of 100 cells per well in a 6-well plate pre-seed with a Swiss 3T3 fibroblast feeder layer, followed by 10 Containing% FBS, 5 μg / ml insulin, 5 μg / ml transferrin, 400 ng / ml hydrocortisone, ^10-9 M cholera toxin, 10 ng / ml EGF, 10 units / ml penicillin and 10 μg / ml streptomycin Incubated in DMEM and Ham's F12 medium (3: 1 mixture). After 12 days of culture, cells were immobilized with 4% formaldehyde and stained with a mixture of 1% Nile Blue and 1% Rhodamine B. Colonies larger than 1 mm in diameter (containing at least about 32274 cells per colony) were counted. CFE values were determined from the averages obtained from each individual experiment using the following formula: Colony Formation Efficiency (%) = (Colonies Number / Seeding Cells) × 100.

Experiment result

Adult rabbit Corneal smoke  In the epithelium SP  Identification of Cells

According to the prior art, SP cells sorted by Hoechst 33342 stain release ability were identified as CD34-negative hematopoietic stem cells [1, 2]. First, the validity of this SP cell sorting method was confirmed using fresh rabbit bone marrow cells (FIG. 1A). SP cells designated by verapamil-sensitive disappearance of unique tails of low Hoechst 33342 Blue / Red fluorescence were successfully identified with an incidence of 1.67 ± 0.76% (n = 5) of bone marrow cells. Using FACS settings for the sorting of rabbit bone marrow SP cells, SP cells were sorted from fresh corneal marginal epithelial cells but SP cells were not detected from corneal epithelial cells (FIG. 1B). The proportion of SP cells in the total epithelial cells of adult rabbit corneal margin was 0.54-0.92 and the mean was 0.73 ± 0.14% (n = 8). The results of the presence of verapamil-sensitive SP cells and their absence in the cornea in the cornea margin obtained through this experiment, according to the conventionally reported fact [20], corneal epithelial stem cells in which SP cells are present in the cornea margin Indicates that

Corneal smoke SP  Cells Corneal smoke MP  Smaller than cells and corneal epithelial cells

Stem cells identified in various tissues are smaller than differentiated cells because stem cells maintain a more primitive cytoplasm and a larger nuclear-to-cytoplasmic ratio. The size distribution of total corneal epithelial cells, total central corneal epithelial cells, corneal lead SP cells and corneal lead MP cells was compared with a forward scatter (FSC) variable [9,28] indicating the cell size (FIG. 1C) . Corneal smoked SP cells were the smallest and showed an acute peak (peak value 200 FSC). However, the size distribution of corneal lead MP cells and corneal epithelial cells was relatively wide. The mean size of cells increased in order of corneal margin SP, corneal margin MP and central corneal epithelial cells. Corneal lead SP cells were the smallest when compared with corneal lead MP cells and corneal epithelial cells, and the results are similar to previous reports on cell size differences between human corneal basal epithelial cells and central corneal basal epithelial cells [28]. Do.

fresh Corneal smoke SP  Cells are undifferentiated and stationary in the cell cycle

If the rabbit corneal lead SP cells isolated in the present invention are corneal epithelial stem cells, these SP cells will exhibit a stationary cell cycle immediately after they have been separated from the tissue, as opposed to active cell proliferation of TA cells [17]. Ki67 [29] and cyclin B1 [30] are known to be expressed in actively cycling corneal epithelial cells. RT-PCR analysis was performed using RNAs isolated from corneal lead SP cells, corneal lead MP cells and central corneal epithelial cells using primers for Ki67 and cyclin B1 (FIG. 2). Corneal lead SP cells did not express Ki67 and cyclin B1. Corneal lead MP cells and central corneal epithelial cells both expressed Ki67 and cyclin B1. Based on the lack of expression of Ki67 and cyclin B1, it was concluded that corneal lead SP cells in the present invention exhibit a stationary cell cycle.

Stem cells are generally poor in intercellular connectivity or adhesion observed in differentiated epithelial cells. In order to determine the degree of differentiation of corneal lead SP cells, RT-PCR was performed using primers for Cornexin 43 and keratin 12 [21,31,32], which are markers of differentiated corneal epithelial cells (FIG. 2). Corneal lead SP cells did not express differentiation-related markers, but both corneal lead MP cells and central corneal epithelial cells expressed both markers. Therefore, it can be seen that the corneal lead SP cells of the present invention are stationary and undifferentiated in the cell cycle, and that corneal lead MP cells and central corneal epithelial cells are composed of TA cells and actively differentiated cells. have.

Corneal smoke SP  Regeneration of Multilayer Corneal Epithelial-like Structure Using Cells

Rabbit corneal lead SP cells identified in the present invention are small, undifferentiated and stationary in the cell cycle. If the SP cells are stationary and corneal epithelial stem cells, they will show an epithelial phenotype under appropriate conditions. We tested whether corneal lead SP cells regenerate corneal-like structures in semi-dry 3-D cell cultures on collagen gels embedded with Swiss 3T3 fibroblasts and in subcutaneous transplantation experiments on nude mice. 3a). Of was successfully reproduced in the similar epithelium, the epithelium of the base layer and the initial low-rise (suprabasal layer) was rich in the positive cell to p63 (Fig. 3b), p63 is a corneal epithelial stem cell - each vague SP cells in vitro cornea from Marker [33] and marker of corneal epithelial TA cells cultured in vitro [34]. Corneal lead MP cells remaining after sorting of SP cells (presumably containing rapidly dividing TA cells) showed superior corneal regeneration ability than corneal lead SP cells alone. Corneal lead MP cells, including actively cycling cells, were effective at regenerating corneal structures during the 5-week 3-D culture period. The best corneal-like equivalent in terms of shape as well as the basal layer pattern of p63 expression similar to the in vivo corneal marginal base layer was achieved in semi-dry 3-D cultures of corneal marginal WP cells (including both SP cells and MP cells). SP cells exhibit a stationary cell cycle immediately after separation from corneal margins, but can activate cell proliferation and regenerate corneal-like epithelium in the semi-dry 3-D cell culture conditions of the present invention.

Corneal regeneration ability was assessed by subcutaneous transplantation of 3-D culture on the back of nude mice (FIG. 3C). Corneal lead SP cells regenerated stratified epithelium, but corneal lead WP cells regenerated tissue similar to the palisade of the cornea lead vogot, where p63 positive cells are very abundant in the basal layer. (FIGS. 3C-3D). Corneal lead MP cells partially regenerated the corneal lead-like structure, and corneal WP cells regenerated corneal-like tissue without the Bot-like structure of the book structure (FIGS. 3C-3D). Thus, corneal lead SP cells can regenerate corneal-like epithelium in vivo , and in order to have their corneal specificity, additional help or direct cell contact of TA cells, which clearly manifests corneal epithelial properties, proliferative capacity or differentiated corneal epithelial cells -It can be seen that the mediation instructions are required.

Central cornea  1 day after wounding Corneal smoke SP  cell Popular  A temporary rise

LRCs are predominantly located in the basal layer of the corneal margin epithelium and are known to proliferate within 24 hours, stimulated by the central corneal wound cycle [18]. We studied whether the central corneal wound cycle can control the population size of corneal lead SP cells (FIG. 4). Alkali-burn in the central cornea allowed for complete removal of the epithelial sheet of the central cornea and partial damage to the upper layer of corneal parenchyma (FIG. 4A, day 0). One day after the wound cycle, the wounded central cornea was barely covered by corneal epithelial cells. On day 5, regenerated corneal epithelium containing 4-5 cell layers was observed, and the opacity of the parenchyma observed on day 0 was somewhat alleviated. Complete recovery from alkali-burn as assessed by corneal clarity was not achieved until 16 days after the wound cycle, and the wounded parenchyma did not fully recover (as assessed by insufficient migration of keratinocytes).

The corneal margin area, which was not directly damaged by alkaline burn, showed no significant change in the epithelium except for the slightly thickened epithelium. In contrast, corneal stroma with only corneal vascular supply showed transient invasion of foreign cells presumed to be carried through corneal blood supply after the wound cycle (FIG. 4A). During the 5 days of corneal recovery after alkaline burn wound cycle, no invasion of conjunctival goblet cells was observed.

The effect of central corneal surface damage on the population size of corneal lead SP cells was tested by Hoechst 33342 emission analysis at different time periods after an alkaline burn wound in the central cornea. Population of corneal lead SP cells increased approximately 5 fold on day 1 after the wound cycle, which was downregulated at rest on day 2 (FIG. 4B). The transient increase of SP cells in response to surface loss, such as the central corneal wound cycle, suggests that SP cells function functionally as stem cells in the corneal epithelium, and these results suggest that cell cycle entry of LRCs along the central corneal wound cycle. Similar to the conventional report for [19].

Corneal smoke Rising SP Cell Population Cornea Precedes CFE elevation of MP cells and subsequent corneal WP cells

A temporary 5-fold increase in corneal lead SP cells on day 1 after the wound cycle contributes to an increase in TA cells in the cornea followed by TA cells in the cornea. Corneal lead SP cells, corneal lead MP cells, or corneal WP cells were isolated at 0, 1, 2 and 5 days after wounding and the CFEs of these cells were compared (FIG. 4C). Corneal smoked SP cells isolated immediately after the wound or the wound cycle did not form colonies efficiently under CFE conditions of 12-day culture. CFE (29.67 ± 3.93%) of corneal lead MP cells at day 0 immediately after the wound cycle was lower than CFE (40.83 ± 4.07%) of corneal WP cells, and this result was due to the residual cornea remaining after the central corneal wound cycle. It seems to be.

One day after the wound cycle in which corneal SP cell populations spiked temporarily, corneal lead SP cells formed colonies with 8% efficiency. However, the CFEs of corneal lead MP cells and corneal WP cells decreased compared to day 0 at the same time point (FIG. 4C), indicating that TA cells rapidly laterally migrate from the corneal margin to the peripheral cornea and central cornea. This may be due to a lot of differentiation on the wound cornea surface.

At day 2 of the wound cycle, CFE of corneal lead MP cells increased 1.67 times compared to day 0, and this increase was maintained until day 5 of wound cycle (FIG. 4C). CFE of corneal WP cells was very large at day 5. This large increase in CFE in the cornea is thought to be due to the temporary increase in corneal lead SP cells on day 1 of the wound cycle.

Corneal smoke Colony appearance of SP cells or bone marrow SP cells, each vaguely Clearly different compared to the appearance of MP cells

Corneal smoked SP cells isolated from resting rabbit corneas did not efficiently produce colonies under epithelial CFE assay conditions (FIG. 4C). To further investigate the culture preference of corneal lead SP cells, SP cells and MP cells isolated from bone marrow or corneal lead were cultured under hematopoietic colony formation assay conditions, and their colony forming ability and colony shape were compared (FIG. 5). . Bone marrow SP cells showed 0.4% CFU, 7 times larger than bone marrow MP cells, and formed colonies in methylcellulose in dispersed hematopoietic form. In contrast, corneal lead SP cells and corneal lead MP cells did not form colonies in methylcellulose, but formed colonies in epithelial shape on the surface of the culture dish. The CFU of corneal lead SP cells was about 0.28%, which is higher than that of corneal lead MP cells. These results are in contrast to the CFE results seen in FIG. 4. The colonies of corneal lead SP cells were true epithelium in terms of shape and differed significantly from the shape of corneal lead MP cells.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Reference

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1A-1C show the flow cytometry results of rabbit bone marrow cells, corneal marginal epithelial cells and central corneal epithelial cells. Rabbit bone marrow cells harvested from femur and tibia and epithelial cells isolated from corneal margin and central cornea were stained with Paste 33342 stain and analyzed by FACS. In FIG. 1A, verapamil-sensitive SP cells were detected in rabbit bone marrow cells with an incidence of 1.67% ± 0.76 (n = 5) on average. In FIG. 1B, verapamil-sensitive SP cells were detected in corneal marginal epithelial cells with an incidence of 0.76% ± 0.14 (n = 8), but no SP cells were detected in central corneal epithelial cells. In FIG. 1C, the size distributions of total corneal marginal epithelial cells, total central corneal epithelial cells, corneal lead SP cells and corneal lead MP cells were compared with FCS variables.

Figure 2 is a photograph showing the results of RT-PCR analysis of rabbit corneal lead SP cells, corneal lead MP cells and central corneal epithelial cells. Expected sizes are Cyclin B1 (275 bp), Ki67 (437 bp), Connexin 43 (538 bp) and Keratin 12 (542 bp). 100 bp DNA ladder was loaded into the first lane and β-actin (125 bp) was used as internal control.

3A-3D show the regeneration of multilayer corneal epithelial-like structures by semi-dry 3-D culture and nude mouse transplantation. 3A is a schematic showing the process of semi-dry 3-D culture and nude mouse transplantation experiments. In FIG. 3B, cross-sections of corneal lead SP cells, corneal lead MP cells and corneal lead WP cells cultured with semi-dry 3-D cells on collagen-keratoparenchymal equivalents were stained with H & E and immunohistochemically Stained. Tissues were stained with H & E two weeks after implantation into the back of nude mice (FIG. 3C) and immunohistochemically with anti-p63 antibodies (FIG. 3D). Dotted line: book structure of Bogot-like structure formed from corneal marginal WP cells; Asterisk: p63 positive cells in early low cell layer; Arrow: p63 positive cells in basal cell layer (B, 200 magnification; C, 100 magnification; D, 200 magnification).

4A-4C show transiently increased corneal lead SP cell population after central corneal wound cycle. Alkaline burns were applied to the rabbit central cornea (6 mm) and the eyes separated on days 0, 1, 2 and 5. Figure 4a is a photograph showing the histological examination results of the central cornea and corneal margin after the wound cycle. Complete loss of corneal epithelium and corneal parenchyma was detected immediately after wounding (day 0). The migration of epithelial cells to the central corneal surface was detected on day 1 and 4-5 epithelial layers were detected on day 5. No significant change in corneal margin epithelium was observed during wound healing, but cell invasion was observed in corneal marginal parenchyma (arrow) on day 1. 4B is a photograph showing up-regulation of transient but distinct corneal marginal SP cell population after central corneal wound cycle. On day 1 verapamil-sensitive SP cells were up-regulated about 5 fold compared to woundless conditions. 4C compares CFE of corneal lead SP cells, corneal lead MP cells and corneal WP cells. Cells were incubated for 12 days for CFE analysis and mean values are indicated by bars. CC: central cornea; PC: peripheral cornea; L: Corneal smoke.

Figures 5a-5b is a comparison of CFU analysis and colony shape of SP cells and MP cells of bone marrow or cornea margin. Cells were seeded in methylcellulose containing CFU assay medium and then cultured for 12 days. Bone marrow-derived cells formed colonies in methylcellulose, but corneal lead-derived cells formed epithelium-like colonies on the surface of the dish. Colony numbers are shown as bars (FIG. 5A) and shapes are shown in FIG. 5B. BM: bone marrow; Lim: Corneal smoke.

Claims (2)

The corneal margin SP (side population) cells are cultured in a medium comprising (i) stem cell factor, (ii) granulocyte-macrophage colony-stimulating factor (GM-CSF) and (iii) interleukin-3 (IL-3). Culture method of corneal lead SP cells comprising the step. The method of culturing corneal lead SP cells according to claim 1, wherein the medium further comprises serum.

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