Functionally improved epithelial stem cells and use therof

Abstract

The present invention relates to functionally improved epithelial stem cells. The epithelial stem cells with improved functionality by a method of the present invention are prepared without genetic modification, thus being less likely to cause side effects due to DNA delivery / modification, are derived from the amniotic membrane in which no tissue-compatible antigen (factor) is expressed, thus having no rejection, and exhibit improved cell migration and would healing effects due to the improved functionality. Therefore, the epithelial stem cells can be used for the regeneration of various tissues, including the cornea, amniotic membrane, skin, and the like, and particularly can be used for the prevention or treatment of premature rupture of the amniotic membrane.

Description

Function-enhancing epithelial stem cells and their uses

[0001] The present invention relates to epithelial stem cells with improved function, and more particularly, to the use of cells expressing a specific marker among epithelial stem cells for cell therapy.

[0002] Wound healing is typically a complex process involving three phases: inflammation, proliferation, and remodeling. The first phase involves clotting to achieve hemostasis, the recruitment of neutrophils to destroy bacteria and necrotic tissue, and the subsequent recruitment of macrophages. During the second phase, angiogenesis occurs, with endothelial cells migrating to the wound site. Simultaneously, fibroblasts migrate to the wound site and help produce granulation tissue. This granulation tissue formation facilitates reepithelialization. During the final phase, collagen production and destruction balance out, and the healed wound slowly remodels to achieve maximum strength. Wound healing can be delayed or impaired if any of these processes fail to function properly, potentially leading to a chronic state. Chronic wounds are defined as open skin wounds requiring healing for more than eight weeks. Impaired healing is often associated with diabetic complications, which can lead to serious outcomes such as high rates of limb amputation and even death. Approximately 15% of diabetic patients suffer from chronic, non-healing wounds. Therefore, wounds are a significant issue both personally and socially, and early and prompt treatment can reduce the risk of secondary infections.

[0003] Meanwhile, the human fetal membrane is composed of the amnion and the chorion. The amnion is a 0.02 to 0.5 mm thick placental membrane with no nerves, lymphatic vessels, or blood vessels, and is located at the innermost part of the placenta. It is composed of five layers: the epithelial layer, the basal layer, the dense layer, the fibroblast layer, and the spongy layer. The amnion surrounds the fetus and acts as a barrier to protect the fetus from various infections and immune responses from the mother. In humans, when the amnion is first formed, the amnion and the fetus are in contact, but after 4-5 weeks of pregnancy, amniotic fluid begins to fill it. As the amniotic fluid fills the amnion, it stretches and eventually comes into contact with the chorion on the outside of the amnion. The amniotic fluid allows the fetus to move freely inside the amnion and cushions external shocks. In addition, the amniotic membrane is receiving attention as an allogeneic graft stem cell therapy because it contains a large number of stem cells and does not express histocompatibility antigens (factors) due to its shared developmental origin with the fetus.

[0004] Preterm premature rupture of membranes (PPROM) is a serious pregnancy complication that occurs when the amniotic membrane surrounding the fetus ruptures during pregnancy, allowing amniotic fluid to leak, leading to infection and premature birth. PPROM is a major cause of perinatal complications and neonatal morbidity and mortality. It is known that PPROM is responsible for 50% of neonatal deaths and 30-40% of premature births, and in particular, 30-50% of PPROM occurs before 37 weeks of pregnancy. Generally, if PPROM occurs before 24-25 weeks of pregnancy, the fetus has difficulty surviving, making miscarriage easy. This is because PPROM causes a serious increase in the risk of intrauterine infection and developmental disorders of the fetal lung system.

[0005] An object of the present invention is to provide epithelial stem cells expressing a specific marker.

[0006] In addition, it is an object of the present invention to provide a cell therapy composition for wound treatment.

[0007] In addition, it is an object of the present invention to provide a pharmaceutical composition for wound treatment.

[0008] In addition, an object of the present invention is to provide a pharmaceutical composition for promoting wound healing.

[0009] In addition, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating preterm premature rupture of membranes (PPROM).

[0010] In addition, an object of the present invention is to provide a method for producing functionally enhanced amniotic epithelial stem cells.

[0011] It is also an object of the present invention to provide a method for treating a wound.

[0012] In addition, it is an object of the present invention to provide a method for preventing or treating preterm premature rupture of membranes (PPROM).

[0013] To achieve the above purpose, the present invention provides epithelial stem cells expressing CD324 marker and SSEA-4 marker.

[0014] In addition, the present invention provides a cell therapeutic agent composition for wound treatment comprising the epithelial stem cells.

[0015] In addition, the present invention provides a pharmaceutical composition for wound treatment comprising the cell therapeutic agent and a carrier.

[0016] In addition, the present invention provides a pharmaceutical composition for promoting wound healing comprising the epithelial stem cells.

[0017] In addition, the present invention provides a pharmaceutical composition for preventing or treating premature rupture of membranes, comprising the epithelial stem cells or cell therapeutic agent.

[0018] In addition, the present invention provides a method for producing functionally enhanced amniotic epithelial stem cells.

[0019] In addition, the present invention provides a method for treating a wound, comprising the step of administering to a subject epithelial stem cells expressing a CD324 marker and a SSEA-4 marker.

[0020] In addition, the present invention provides a method for preventing or treating preterm premature rupture of membranes (PPROM), comprising a step of administering to a subject epithelial stem cells expressing CD324 marker and SSEA-4 marker.

[0021] The epithelial stem cells with improved functionality by the method of the present invention are manufactured without genetic modification, so there is less possibility of side effects due to DNA transfer / modification, and since they are derived from amniotic membranes that do not express histocompatibility antigens (factors), there is no rejection reaction, and since cell mobility and wound healing effects are improved due to improved functionality, they can be used for the regeneration of various tissues, including cornea, amniotic membrane, and skin, and are particularly effective in preventing or treating premature rupture of membranes.

[0022] Figure 1 is a diagram showing the process of extracting amniotic epithelial stem cells from amniotic tissue.

[0023] Figure 2 is a diagram showing the yield of passage 1 amniotic epithelial stem cells selected using the CD324 marker.

[0024] Figure 3 shows the results of immunocytochemical staining to confirm the expression of stem cell markers in cells before and after cell selection. Figure 4(A) shows the results of confirming CD324 expression, Figure 4(B) shows the results of confirming OCT3 / 4 and CD324 expression, Figure 4(C) shows the results of confirming Nanog and CD324 expression, and Figure 4(D) shows the results of confirming SOX-2 and CD324 expression.

[0025] Figure 4 shows the results of immunocytochemical staining to confirm the expression of stem cell markers in cells before and after cell selection. Figure 5 (A) shows the results of confirming CD90 and CD324 expression, Figure 5 (B) shows the results of confirming CD105 and CD324 expression, Figure 5 (C) shows the results of confirming CD19 and CD324 expression, and Figure 5 (D) shows the results of confirming CD34 and CD324 expression.

[0026] Figure 5 is a diagram showing the yield of passage 2 amniotic epithelial stem cells selected using the SSEA-4 marker.

[0027] Figure 6 is a diagram showing the yield of passage 2 amniotic epithelial stem cells selected using the CD324 marker.

[0028] Figure 7 is a diagram showing the expression of CD324 and SSEA-4 in functionally enhanced amniotic epithelial stem cells selected with the CD324 marker, confirmed by immunofluorescence staining.

[0029] Figure 8 is a diagram analyzing the cell mobility of SSEA-4 and CD324 positive functionally enhanced amniotic epithelial stem cells.

[0030] Figure 9 is a diagram confirming the wound healing effect of functionally enhanced amniotic epithelial stem cells in a premature rupture of membranes model.

[0031] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments of the present invention. However, the following exemplary embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0032] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0033] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited herein as references are incorporated herein by reference.

[0034] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise stated.

[0035]

[0036] In one aspect, the present invention relates to epithelial stem cells expressing CD324 marker and SSEA-4 marker.

[0037] In one embodiment, the epithelial stem cells may additionally express CD105, CD90, OCT3 / 4, Nanog or SOX-2 markers.

[0038] In one embodiment, the epithelial stem cell may be an amniotic epithelial stem cell derived from the amniotic membrane.

[0039] In one embodiment, amniotic epithelial stem cells derived from the amniotic membrane may be epithelial cells and mesenchymal cells having stem cell properties, and are capable of differentiating into endoderm, mesoderm, and ectoderm (3 germ layers).

[0040] Stem cells may be adult stem cells, mesenchymal stem cells (MSCs), or have properties of mesenchymal stem cells (MSCs).

[0041] The term "stem cell" used in the present invention refers to undifferentiated cells capable of differentiation into various cells that constitute biological tissues and capable of unlimited regeneration to form specialized cells of tissues and organs. Stem cells are pluripotent or multipotent cells capable of development. Stem cells proliferate into mature and complete cells of tissues.

[0042] The term "mesenchymal stem cell" used in the present invention refers to an undifferentiated cell having multipotency derived from an adult cell of a mammal, including a human, preferably a human, and means a stem cell having multipotency capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. The mesenchymal stem cell can be derived from various adult cells, such as bone marrow, blood, brain, skin, fat (i.e., adipose tissue or adipocytes), umbilical cord blood, and umbilical cord Wharton's jelly, for example.

[0043] The term "differentiation" used in the present invention refers to a phenomenon in which less specialized cells develop into specific cells, and the size, shape, membrane potential, metabolic activity, and response to signals of the cell change into a specific type of cell. Differentiation is defined as a qualitative difference occurring between parts of a biological system that were initially nearly homogeneous, or a state in which the system is divided into qualitatively distinguishable subsystems as a result. In particular, differentiation of stem cells refers to a phenomenon in which stem cells develop in a direction into cells with specific functions.

[0044] The term "expression" as used herein generally refers to the cellular process by which a biologically active polypeptide is generated from a DNA sequence and exhibits biological activity in a cell. In this sense, gene expression encompasses not only transcription and translation processes, but also post-transcriptional and post-translational processes that can affect the biological activity of a gene or gene product. These processes include, but are not limited to, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of the polypeptide.

[0045] In the present invention, the expression can be confirmed by measuring the expression level of a gene or mRNA using a nucleic acid sequence, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer pair and a probe that specifically recognize a fragment of the nucleic acid sequence and the complementary sequence, a polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip method, and using an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognizes the full-length protein or a fragment thereof, a Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), The expression level of the protein can be measured and confirmed using radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray methods.

[0046] In one embodiment, the epithelial stem cells may have increased mobility.

[0047] In one aspect, the present invention relates to a cell therapy composition for wound treatment comprising the epithelial stem cells of the present invention.

[0048] In one embodiment, the composition may further comprise HGF or bFGF.

[0049] In one embodiment, the epithelial stem cells may be 3D cultured stem cells.

[0050] In one embodiment, the cell therapy composition can be stored frozen and used for wound healing.

[0051] In one embodiment, the cell therapy agent may be administered to the human body through any common route as long as it can reach the target tissue, and may be administered parenterally or orally, and when administered orally, the cells may be incorporated into a graft material or the like.

[0052] In one embodiment, the cell therapy agent can be used for corneal regeneration or skin regeneration.

[0053] In one embodiment, the cell therapy agent may be for allogeneic grafting.

[0054] The term "cell therapeutic agent" used in the present invention refers to a therapeutic agent used for tissue regeneration treatment by proliferating and selecting living autologous, allogenic, or xenogenic epithelial stem cells in vitro and introducing them into the body to restore the tissue and function of cells.

[0055] In one aspect, the present invention relates to a pharmaceutical composition for wound treatment, comprising a therapeutically effective amount of a cell therapeutic agent of the present invention and a pharmaceutically acceptable carrier.

[0056] In one embodiment, the carrier may be selected from the group consisting of ointments, gels, gauze, bandages, bandages, films, adhesive patches and non-adhesive patches.

[0057] In one aspect, the present invention relates to a pharmaceutical composition for promoting wound healing, comprising the epithelial stem cells of the present invention.

[0058] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating preterm premature rupture of membranes (PPROM), comprising the epithelial stem cells of the present invention or the cell therapeutic agent of the present invention.

[0059] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat premature rupture of membranes at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the subject's health condition, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0060] The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The term "pharmaceutically acceptable" as used herein means that the composition exhibits non-toxic properties to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and for example, a compound, saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main use form such as an aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component using an appropriate method in the art.

[0061] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group consisting of oral dosage forms, topical preparations, suppositories, sterile injectable solutions, and sprays.

[0062] The composition of the present invention may also include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and for example, a compound, saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main use form such as an aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component using an appropriate method in the art.

[0063] In addition, the composition of the present invention may contain one or more active ingredients exhibiting the same or similar function.

[0064] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0065] The composition of the present invention can be administered non-orally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer it once or several times a day.

[0066] In one aspect, the present invention relates to a transplant material for amniotic membrane regeneration comprising the epithelial stem cells of the present invention or the cell therapeutic agent composition of the present invention.

[0067] In one embodiment, the graft material may be a surgical implant, medical device, or medical apparatus such as a film, membrane, sheet, rod, screw, anchor, pin, implant, stent, surgical suture, scaffold for tissue regeneration, bio-nano fiber, hydrogel, bio-sponge, bone plate, and bone graft.

[0068] In one embodiment, if the graft material is a hydrogel, it can be administered parenterally to the amniotic membrane to promote regeneration of amniotic tissue.

[0069] In one embodiment, the graft material may further comprise a biodegradable polymer.

[0070] In one embodiment, the graft material may be a composite scaffold for tissue engineering, and the composite scaffold for tissue engineering may include a stem cell of the present invention or a cell therapeutic agent composition of the present invention in a scaffold made by molding a biodegradable polymer.

[0071] In one embodiment, the graft material may be a graft material in which the stem cell or cell therapeutic agent of the present invention is inoculated into a composite support for tissue engineering.

[0072] In one embodiment, when the graft material is a hydrogel, the polymer forming the hydrogel may be polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyacrylic acid (PAA), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), gelatin, hyaluronic acid, alginate, carrageenan, chitosan, hydroxyalkylcellulose, alkylcellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylate, polyvinyl chloride, or maleic anhydride / vinyl ether.

[0073] The term "biodegradable polymer" used in the present invention refers to a polymer that is slowly and spontaneously decomposed in a living body after a certain period of time, and has one or more of the following properties: biocompatibility, blood compatibility, anti-calcification properties, cellular nutritional properties, and intercellular matrix formation ability. The type of such biodegradable polymer is not particularly limited in the present invention, but representative examples thereof include fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, poly(glycolic acid), PGA, poly(lactic-co-glycolic acid), PLGA, poly-ε-(caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer, copolymers thereof, and mixtures thereof. The above composite support can be manufactured by molding a biodegradable polymer according to a conventionally known method, for example, a solvent-casting and particle-leaching technique, a gas forming technique, a fiber extrusion and fabric forming process for manufacturing a polymer mesh by forming polymer fibers into a nonwoven fabric, a thermally induced phase separation technique, an emulsion freeze drying method, a high pressure gas expansion method, etc.

[0074] In one aspect, the present invention relates to a method for producing functionally enhanced amniotic epithelial stem cells, comprising the steps of: 1) isolating cells from isolated amniotic tissue; and 2) isolating CD324 positive cells from the cells.

[0075] In one embodiment, the step of isolating the cells may further include a step of subculturing the isolated cells.

[0076] In one embodiment, the subculturing may be performed 1 to 10 times. Preferably, it may be performed 1 to 5 times, more preferably, it may be performed 2 to 4 times, and even more preferably, it may be performed 2 times, but is not limited thereto.

[0077] In one embodiment, the method may further include a step of isolating and subculturing SSEA-4 positive cells after step 1).

[0078] In one embodiment, the CD324 positive cells of step 2) are characterized in that they are selected from SSEA-4 positive cells.

[0079] In one embodiment, the functional improvement is characterized by at least one selected from the group consisting of mobility and wound healing ability of amniotic epithelial stem cells.

[0080] In one aspect, the present invention relates to a method for treating a wound, comprising the step of administering to a subject epithelial stem cells expressing a CD324 marker and a SSEA-4 marker.

[0081] In one aspect, the present invention relates to a method for preventing or treating preterm premature rupture of membranes (PPROM), comprising the step of administering to a subject epithelial stem cells expressing CD324 marker and SSEA-4 marker.

[0082] In one embodiment, the epithelial stem cells may additionally express CD105, CD90, OCT3 / 4, Nanog or SOX-2 markers.

[0083] In one embodiment, the epithelial stem cell may be an amniotic epithelial stem cell derived from the amniotic membrane.

[0084] In the method for treating a wound of the present invention or the method for preventing or treating premature rupture of membranes, the “subject” may be administered to an “subject” that requires wound treatment or prevention or treatment of premature rupture of membranes, or has developed or is likely to develop a wound or premature rupture of membranes, and the “subject” may mean any animal including a human.

[0085] In the method for treating a wound of the present invention or the method for preventing or treating premature rupture of membranes, the specific description is the same as above.

[0086]

[0087] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0088]

[0089] Example 1. Production of functionally enhanced amniotic epithelial stem cells derived from amniotic tissue.

[0090] Human amniotic epithelial stem cells (AESCs) expressing stem cell markers SSEA-4, SOX-2, Nanog, and / or OCT-3 / 4 were extracted by dissociating amniotic tissue isolated from the placenta discarded during the birth process into single cells using trypsin and then passaged (Fig. 1). As a result of sorting CD324 positive cells using a FACS ARIA III instrument from passage 1 amniotic epithelial stem cells, 2.0 × 10 6Approximately 80% or 90% of the cells were obtained (Fig. 2). The expression of stem cell markers in the cells before and after cell sorting was confirmed by immunocytochemical staining, and the expression of the same markers was confirmed to be maintained (Figs. 3 and 4). In addition, when the passage 2 amniotic epithelial stem cells were sorted for CD324 positive cells using a FACS ARIA III device, the yield of SSEA-4 positive cells was approximately 71%, and the yield of CD324 was approximately 30% (Figs. 5 and 6). The expression of the SSEA-4 marker was confirmed by immunocytochemical staining in the CD324 positive functional amniotic epithelial stem cells with enhanced epithelial stem cell characteristics obtained in this way was found to be strong in the expression of both CD324 and SSEA-4 (Fig. 7).

[0091]

[0092] Example 2. Mobility analysis of functionally enhanced amniotic epithelial stem cells derived from amniotic membrane tissue

[0093] To evaluate the mobility of the functionally enhanced amniotic epithelial stem cells manufactured in Example 1 above, 2.0×10 4 After dispensing the amniotic epithelial stem cells with enhanced function and the amniotic epithelial stem cells without separation of cells with markers (control group), the cells were cultured to grow on all surfaces of the plate, and the silicone in the center was removed and cultured for 2 days to analyze cell mobility. In addition, to evaluate cell mobility by another method, the Transwell Kit (Catalog. 353097) was placed in a 24-well plate and 2.0 × 10 4 After seeding the cells (approximately 50% of the bottom surface), they were cultured for one day and the cells that migrated and attached to the bottom layer of the Transwell Kit were stained and counted using 0.1% crystal violet dye.

[0094] As a result, it was found that the mobility of the functionally enhanced amniotic epithelial stem cells of the present invention, which include cells isolated using a specific marker, was significantly higher than that of amniotic epithelial stem cells (Fig. 8).

[0095]

[0096] Example 3. Analysis of the wound healing effect of amniotic epithelial stem cells derived from amniotic membrane tissue with enhanced function.

[0097] In order to confirm the treatment effect of premature rupture of membranes through the wound healing effect of the functionally enhanced amniotic epithelial stem cells manufactured in Example 1, circular perforated amniotic membranes were manufactured to mimic premature rupture of membranes in vitro, and the natural wound healing process was compared when functionally enhanced amniotic epithelial stem cells were used. Specifically, amniotic tissue was isolated and thoroughly washed with sterile PBS to eliminate contamination from other tissues. The isolated amniotic membrane was then transferred to a sterile glass culture dish and sterile saline solution was added to prevent drying. The amniotic membrane was evenly spread with tweezers, and a 5 mm diameter biopsy punch was held vertically and pressed down on the amniotic membrane from top to bottom to remove dozens of 5 mm diameter amniotic membrane tissues. The removed amniotic membrane tissue was then perforated in the center using a 1 mm diameter biopsy punch to produce 5 mm circular perforated amniotic membrane tissues with 1 mm diameter perforations. Each circular perforated amniotic membrane tissue was placed in a 24-well plate with one tissue per well, and the tissue was fixed in a well-spread state without being wrinkled or folded, and cultured. After that, the functionally enhanced amniotic epithelial stem cells manufactured in Example 1 were added to the untreated (0), 1 × 10 4 Dog, 2×10 4 Dog and 5×10 4 After each treatment, the regeneration of amniotic membrane tissue was confirmed over time. At this time, 5×10 CD105 and SSEA-4 positive cells were used as a control group. 4After treatment with a dog, regeneration of amniotic membrane tissue over time was confirmed. The CD105 and SSEA-4 positive cells were obtained using the same method as Example 1, and in the method, cells positive for CD105 were isolated instead of CD324.

[0098] As a result, it was found that the functionally enhanced amniotic epithelial stem cells according to the present invention increased the regeneration of amniotic tissue in a manner dependent on the number of treated cells (Fig. 9). In contrast, in the case of the comparative CD105 and SSEA-4 positive cells, the cells were well cultured in the perforated area, but when the functionally enhanced amniotic epithelial stem cells according to the present invention were cultured at 5 × 10 4 The effect of reducing the size of the perforation was minimal compared to the experimental group treated with dogs.

Claims

1. Epithelial stem cells expressing CD324 marker and SSEA-4 marker.

2. In the first paragraph, the epithelial stem cell additionally expresses CD105, CD90, OCT3 / 4, Nanog or SOX-2 markers.

3. In the first paragraph, the epithelial stem cell is an amniotic epithelial stem cell derived from the amniotic membrane.

4. In the first paragraph, the epithelial stem cell is an epithelial stem cell having increased mobility.

5. A cell therapy composition for wound treatment, comprising the epithelial stem cells of paragraph 1.

6. A pharmaceutical composition for wound treatment, comprising a therapeutically effective amount of the cell therapy agent of paragraph 5 and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition for wound treatment in claim 6, wherein the carrier is selected from the group consisting of ointment, gel, gauze, bandage, band, film, adhesive patch and non-adhesive patch.

8. A pharmaceutical composition for promoting wound healing, comprising the epithelial stem cells of paragraph 1.

9. A pharmaceutical composition for preventing or treating preterm premature rupture of membranes (PPROM), comprising the epithelial stem cell of paragraph 1 or the cell therapeutic agent of paragraph 5. 10.1) A step of isolating cells from the isolated amniotic tissue; and 2) A method for producing functionally enhanced amniotic epithelial stem cells, comprising the step of isolating CD324 positive cells from the above cells.

11. A method for producing functionally enhanced amniotic epithelial stem cells, further comprising a step of isolating and subculturing SSEA-4 positive cells after step 1) in the 10th paragraph.

12. A method for producing functionally enhanced amniotic epithelial stem cells, characterized in that the CD324 positive cells of step 2) in paragraph 10 are selected from SSEA-4 positive cells.

13. A method for producing functionally improved amniotic epithelial stem cells, wherein the functionally improved amniotic epithelial stem cells in paragraph 10 are at least one selected from the group consisting of mobility and wound healing ability of amniotic epithelial stem cells.

14. A method for treating a wound, comprising the step of administering to a subject epithelial stem cells expressing CD324 marker and SSEA-4 marker.

15. A method for treating a wound in claim 14, wherein the epithelial stem cells additionally express CD105, CD90, OCT3 / 4, Nanog or SOX-2 markers.

16. A method for treating a wound in claim 14, wherein the epithelial stem cells are amniotic epithelial stem cells derived from the amniotic membrane.

17. A method for preventing or treating preterm premature rupture of membranes (PPROM), comprising the step of administering to a subject epithelial stem cells expressing CD324 marker and SSEA-4 marker.

18. A method for preventing or treating premature rupture of membranes in claim 17, wherein the epithelial stem cells additionally express CD105, CD90, OCT3 / 4, Nanog or SOX-2 markers.

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