WO2022197160A1 - Stem cells derived from intervillous space adjacent to decidua and cellular therapeutic agent for tissue regeneration comprising same - Google Patents
Stem cells derived from intervillous space adjacent to decidua and cellular therapeutic agent for tissue regeneration comprising same Download PDFInfo
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- WO2022197160A1 WO2022197160A1 PCT/KR2022/003849 KR2022003849W WO2022197160A1 WO 2022197160 A1 WO2022197160 A1 WO 2022197160A1 KR 2022003849 W KR2022003849 W KR 2022003849W WO 2022197160 A1 WO2022197160 A1 WO 2022197160A1
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- stem cells
- decidua
- cartilage
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- interchorionic
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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Definitions
- the present invention relates to stem cells derived from interchorionic cavity tissue, preferably from the interchorionic space adjacent to the decidua, and a cell therapy product containing the same.
- Stem cell refers to a cell that has the ability to differentiate into two or more cells while having the ability to self-replicate, totipotent stem cell, pluripotent stem cell, It can be classified as a multipotent (multipotent) stem cell.
- a totipotent stem cell is a cell with pluripotent properties that can develop into a complete individual. Cells up to the 8th cell stage after fertilization of an egg and sperm have these properties. Transplantation can develop into a complete individual.
- Pluripotent stem cells are cells that can develop into various cells and tissues derived from ectoderm, mesoderm, and endoderm. The inner cell mass is located inside the blastocyst that appears 4-5 days after fertilization.
- Multipotent stem cells are stem cells that can differentiate only into cells specific to the tissues and organs that contain these cells.
- Adult bone marrow, skin, blood vessels, muscle, etc. are known as sources of pluripotent stem cells as described above, and these stem cells are rapidly being applied to tissue engineering, gene therapy, and cell therapy fields. It is a situation in which stem cells are obtained and various applications thereof are urgently required.
- the placenta is an organ that develops from the uterine wall when a woman is pregnant, and has a disk-like shape rich in vascular tissues. As scientific research results that lifelong health is determined during the fetal period are reported, the importance of the placenta during pregnancy is being emphasized, and many efforts are being made to clarify the interrelationship between various substances related to it.
- the placenta is composed of various types of cells by age and location, but its application is still very limited.
- placental tissue-derived stem cells are stem cells derived from some tissues of a term placenta obtained during childbirth.
- the placenta is composed of various tissues such as the basement membrane attached to the uterus, the chorionic tissue that mediates the exchange of substances between mother and fetal blood as the central tissue of the placenta, and the chorionic membrane that covers the placenta inside the placenta and surrounds the fetus and the amniotic membrane.
- NAC N-acrtyl-L-cysteine
- bFGF Basic Fibroblast
- stem cells obtained from subdivided placenta and subdivided sites and their effects have not yet been widely reported, regardless of conventional anatomically known methods for distinguishing placenta structures.
- the decidual adjacent interchorionic cavity (CT-V2) was obtained and stem cells were isolated therefrom.
- the isolated decidual-proximal interchorionic space-derived stem cells were obtained from the whole placenta, the entire intervillous space (IVS), and the chorionic plate-adjacent interchorionic space (CT-V1)
- the present invention was completed by confirming that the derived stem cells and CD marker expression patterns were different, and the colony forming ability, proliferative ability, and differentiation ability into cartilage, bone and fat were all significantly different.
- stem cells derived from the interchorionic cavity adjacent to the decidua a cell therapy agent using the same, a composition for tissue regeneration, and a method for separating them.
- the present invention is a stem cell derived from the decidua adjacent interchorionic space, wherein the decidual adjacent interchorionic space is a trophoblast from the decidua adjacent site to the 2/3 point of the entire trophoblast layer, the decidua adjacent villi Provided is a liver-derived stem cell.
- the present invention comprises the steps of: 1) cutting the trophoblast layer from the adjacent decidua to 2/3 of the entire trophoblast layer to obtain a trophoblast adjacent to the decidua; 2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; It provides a method for separating and obtaining stem cells derived from the interchorionic space adjacent to the decidua, comprising a.
- the present invention provides a cell therapy agent for tissue regeneration comprising the stem cells as an active ingredient.
- the present invention also provides a composition for tissue regeneration comprising the stem cells as an active ingredient.
- the present invention provides a tissue regeneration use of stem cells derived from the decidua adjacent to the trophoblast, which is the trophoblast of the region from the region adjacent to the decidua to the 2/3 point of the entire trophoblast layer.
- the present invention provides the use of a cell therapeutic for tissue regeneration of stem cells derived from the decidua adjacent to the trophoblast, which is the trophoblast in the region from the region adjacent to the decidua to the 2/3 point of the entire trophoblast layer.
- the present invention provides a tissue regeneration method, comprising administering to an individual in need thereof, stem cells derived from the decidua adjacent to the trophoblast, which are the trophoblasts of the region from the region adjacent to the decidua to the 2/3 point of the entire trophoblast layer. .
- the decidua-proximal interchorionic space-derived stem cells according to the present invention exhibit homogeneous growth characteristics and excellent proliferation characteristics compared to other placental tissue-derived stem cells, and have remarkably excellent ability to differentiate into cartilage, bone and fat. It can be effectively used for various tissue regeneration treatments that require the regeneration of bone, especially cartilage regeneration and osteoarthritis treatment.
- FIG. 1 is a schematic diagram of the whole placenta, the entire intervillous space (IVS), the chorionic plate adjacent interchorionic space (CT-V1), and the decidual adjacent interchorionic space (CT-V2).
- Figure 2 is after culturing stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2); It is a diagram showing the result of observing the cell morphology under a microscope.
- Figure 3 shows the colony forming performance of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1), and decidual adjacent interchorionic space (CT-V2); It is a diagram showing the confirmed results (*P>0.05, ***P>0.001).
- Figure 5 shows adipocyte differentiation ability of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1), and decidual adjacent interchorionic space (CT-V2); is a diagram showing the results of confirming (*P>0.05, **P>0.01).
- FIG. 6 is a chondrocyte differentiation ability of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2); is a diagram showing the results of confirming (**P>0.01, ***P>0.001).
- FIG. 7 shows the osteocytic differentiation potential of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1), and decidual adjacent interchorionic space (CT-V2); is a diagram showing the results of confirming (*P>0.05, ***P>0.001).
- FIG. 8 is a view showing the cartilage regeneration effect after mixed administration of stem cells derived from the interchorionic space adjacent to the chorionic plate (CT-V1) and the decidua adjacent interchorionic space (CT-V2) with hyaluronic acid (HA) to the rat cartilage defect animal model. It is a figure which shows the evaluation result.
- Figure 9 is a rat cartilage defect animal model after chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2) derived stem cells and hyaluronic acid after mixed administration, the regeneration effect using the ICRS macroscopic score It is a diagram showing the evaluation results (*P>0.01, **P>0.001, ***P>0.001).
- Figure 10 is after mixed administration of stem cells derived from the chorionic interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2) and hyaluronic acid to the rat cartilage defect animal model, H&E, Safranin O and type II collagen staining It is a diagram showing the results of confirming the regeneration effect through
- Figure 11 is after mixed administration of stem cells and hyaluronic acid derived from the chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2) to the rat cartilage defect animal model, Modified O'driscoll score for the regeneration effect It is a diagram showing the results confirmed through (*P>0.01, **P>0.001, ***P>0.001).
- the present invention provides a stem cell derived from the decidual space adjacent to the decidua, a cell therapeutic agent for tissue regeneration comprising the same, and a composition for tissue regeneration.
- the present invention provides stem cells derived from the decidua adjacent interchorionic space, wherein the decidual adjacent interchorionic space is a trophoblast of the entire trophoblast adjacent to the decidua to 2/3 of the entire trophoblast layer. .
- stem cell refers to a cell having the ability to differentiate into two or more different types of cells while having the ability to self-replicate.
- Stem cells may be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells according to their differentiation capacity.
- placenta means an in vivo tissue made for a fetus during pregnancy, and is in the form of a disc having a weight of 500-600 g, a diameter of 15-20 cm, and a thickness of about 2-3 cm.
- One side of the placenta is in contact with the mother and the other side is in contact with the fetus, between which nutrients and oxygen are transferred between the blood vessels of the mother and the fetus.
- the placenta can be largely divided into three layers of amniotic membrane, chorion, and decidua, and more specifically, can be divided into amniotic epithelium, amniotic membrane, chorion, trophoblast, and decidua.
- the intervillous space (IVS) present in the trophoblast layer (Chorion) of the placenta is divided in more detail to obtain the interchorionic space of the trophoblast layer adjacent to the decidua, and stem cells are obtained therefrom. do.
- the 'trophic layer adjacent to the decidua' is a trophic layer located on the decidua side of the total trophoblast. When the entire trophoblast layer is divided into three, it is defined as a continuous region from the adjacent decidua to 2/3 points.
- the region in which villi were excluded from the trophoblast layer adjacent to the decidua was defined as the 'intervilli space adjacent to the decidua,' and in the present invention, it was designated as 'CT-V2'.
- the chorionic plate adjacent interchorionic cavity region is defined as a region excluding the villi in the trophoblast layer, which is a continuous region from the chorionic plate adjacent region to 1/3 point when the entire trophoblast layer is divided into thirds.
- stem cells derived from the decidual interchorionic space have a different CD marker expression pattern compared to stem cells derived from the whole placenta, the entire interchorionic space and the chorionic plate adjacent interchorionic space, and have homogeneous growth characteristics and excellent proliferation. It was confirmed that the ability to differentiate into cartilage, bone and fat was remarkably excellent.
- the decidual adjacent interchorionic space (CT-V2)-derived stem cells of the present invention have negative surface factor expression characteristics for CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4;
- CD49b, CD74 or CD200 may be characterized as having a positive surface factor expression characteristic, preferably one or more selected from the group consisting of CD24, CD104, CD227, Disialogangolioside GD2 and SSEA-4, two or more, Three or more, four or more, and all five surface factors may be all negative, and/or one or more, two or more, or all three surface factors selected from the group consisting of CD49b, CD74 and CD200 may all be positive.
- This surface factor expression characteristic is a unique surface factor expression characteristic of the stem cells derived from the decidua adjacent interchorionic space (CT-V2) of the present invention, which is distinguished from stem cells derived from the whole placenta, the entire interchorionic cavity, and the chorionic plate adjacent interchorionic space. Unlike stem cells derived from the whole placenta, the entire interchorionic cavity and the chorionic plate adjacent interchorionic space, the stem cells of the present invention are newly isolated in that they show high expression of CD49b and CD200 surface factors and negative surface marker expression patterns for CD24 and CD227. It was confirmed that the stem cells were
- the decidual adjacent interchorionic space (CT-V2)-derived stem cells of the present invention CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4 negative surface factor expression; and positive surface factor expression properties for CD44, CD73, CD90, CD105, CD49b, CD74 or CD200, the CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 and SSEA-4 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 surface factors selected from the group consisting of are all negative, and / or CD44, One or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, and 7 surface factors selected from the group consisting of CD73, CD90, CD105, CD49b, CD74 and CD200 may all be positive.
- the stem cells derived from the decidua adjacent interchorionic space may be stem cells derived from parental cells.
- the placenta is a unique tissue that shows the characteristics of the fetus and the mother.
- placental-derived stem cells may have different origins depending on the location where they are obtained, trophoblast-derived stem cells are of maternal origin, villous-derived stem cells are fetal cells, and whole placental-derived stem cells are fetal and maternal. It is known that stem cells originate from both.
- the stem cells derived from the decidua adjacent interchorionic space (CT-V2) of the present invention were confirmed to have a maternal-derived cell chromosome type as a result of chromosomal analysis.
- stem cells derived from the interchorionic space (CT-V2) adjacent to the decidua can be obtained using any method known in the art without limitation, and preferably 1) from the region adjacent to the decidua in the entire trophoblast layer to 2/3 points cutting the phosphorus trophoblast layer to obtain a trophoblast layer adjacent to the decidua; 2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; Characterized in that obtained through; can be obtained through
- the present invention comprises the steps of 1) obtaining a trophoblast adjacent to the decidua by cutting the portion of the trophoblast that is from the portion adjacent to the decidua to the 2/3 point among the entire trophoblast layer; 2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; It provides a method for separating and obtaining stem cells derived from the interchorionic space adjacent to the decidua, comprising a.
- Step 1) is a step of separating the trophoblast adjacent to the decidua out of the entire trophoblast layer separated from the placenta.
- Step 2) is a step of separating the trophoblast adjacent to the decidua out of the entire trophoblast layer separated from the placenta.
- step 2) the villi are removed from the trophoblast layer adjacent to the decidua in which the villi and the interchorionic cavity exist together, and thus only the pure interchorionic cavity is separated.
- the method may further include washing the interchorionic cavity adjacent to the decidua with PBS to remove the remaining blood and blood cells.
- step 3) is a step of obtaining stem cells from the interchorionic space tissue adjacent to the decidua.
- the cells obtained by adding an enzyme solution to the interchorionic space tissue adjacent to the decidua and performing an enzymatic reaction, without using growth factors, are treated with fetal bovine serum and It can be obtained by culturing in an antibiotic-added medium and then recovering.
- the enzymes include trypsin, collagenase, dispase, DNase, RNase, protease, lipase, hyaluronidase and elastase. and the like, but are not limited thereto.
- the collagenase may include collagenase A, I, II, III or IV, and the like.
- the decidual-proximal interchorionic space-derived stem cells isolated by the above method have negative surface factor expression characteristics for CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4; Or CD49b, CD74 or CD200 may be characterized as having a positive surface factor expression characteristic, preferably one or more selected from the group consisting of CD24, CD104, CD227, Disialogangolioside GD2 and SSEA-4, two or more, Three or more, four or more, and five surface factors may all be negative, and/or one or more, two or more, and three surface factors selected from the group consisting of CD49b, CD74 and CD200 may all be positive.
- the stem cells derived from the decidual interchorionic space (CT-V2) according to the present invention can be differentiated into different types of cells, for example, adipocytes, chondrocytes, osteocytes, nerve cells, ligament cells, or tendon cells. (tenocyte) may be differentiated into various types of cells, but is not limited thereto.
- the present invention provides a cell therapy agent for tissue regeneration and a composition for tissue regeneration comprising stem cells derived from the decidua adjacent interchorionic space (CT-V2) as an active ingredient.
- C-V2 decidua adjacent interchorionic space
- the present invention comprises the steps of treating a subject in need thereof with stem cells derived from the chorionic space adjacent to the decidua;
- a tissue regeneration method comprising a
- the interchorionic cavity adjacent to the decidua provides a tissue regeneration method, which is a trophoblast of the entire trophoblast adjacent to the decidua to 2/3 of the total trophoblast.
- differentiation generally refers to a phenomenon in which a relatively simple limit is separated into two or more qualitatively different subsystems. In other words, it means a phenomenon in which cells, tissues, etc. of living things change form or function in order to perform a given task.
- undifferentiated refers to a state in which the above-described differentiation has not occurred and yet contains the characteristics of stem cells.
- the method for differentiating stem cells may be performed according to a conventionally known method, and is not particularly limited.
- a method of culturing the stem cells in a medium containing dexamethasone, indomethacin, insulin and IBMX (3-isobutyl-1-methylxanthine) to differentiate them into adipocytes A method of culturing the stem cells in a medium containing dexamethasone, bone morphogenetic protein 6 (BMP-6), TGF- ⁇ factor beta, ascorbic acid and L-proline to differentiate them into chondrocytes ; It is preferable to use a method for differentiating the stem cells into osteocytes by culturing the stem cells in a medium containing dexamethasone, ascorbic acid, and ⁇ -glycrophosphate ( ⁇ and ascorbic acid-2-phosphate).
- the method for measuring the degree of differentiation of stem cells derived from the decidual adjacent interchorionic cavity tissue differentiated by the above method is not particularly limited thereto, but is a technique known in the art such as flow cytometry, immunocytochemical method, PCR or gene- A method of measuring a change in cell surface label or morphology using an expression profile, a method of examining a morphological change of a cell using an optical microscope or a confocal microscope, a method of measuring a change in a gene expression profile, etc.
- RT-PCR Oil-red O staining
- Safranin O staining Type II collagen immunohistochemical staining
- ALP (alkaline phosphate) staining or Alizarin red S staining
- Alizarin red S staining can be used.
- the term "cellular therapeutic agent” refers to cells and tissues isolated from humans, cultured, and manufactured through special manipulation, and as pharmaceuticals (U.S. FDA regulations) used for the purpose of treatment, diagnosis, and prevention. Through a series of actions, such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways to restore the function of tissues, these cells are used for the purpose of treatment, diagnosis and prevention of diseases medicines used as
- the stem cells derived from the decidua proximal interchorionic cavity tissue of the present invention are various types of tissue or organs in the body that are modulated, enhanced, treated or replaced by engraftment, transplantation, or injection of a desired cell population, for example, a stem cell or differentiated cell population. It can be used for any kind of treatment protocol. Interchorionic cavity tissue-derived stem cells of the present invention can replace or strengthen existing tissues, resulting in new or altered tissues, or can be combined with biological tissues or structures.
- the cell therapy agent of the present invention can be used for regenerating one or more tissues selected from the group consisting of fat, cartilage, bone, nerve, ligament and tendon.
- the cartilage may include, without limitation, free cartilage, fibrocartilage or elastic cartilage, and the cartilage is articular cartilage, ear cartilage, noncartilage, elbow cartilage, meniscus, knee. It may be characterized in that it is selected from the group consisting of cartilage, cost cartilage, ankle cartilage, cartilage cartilage, occipital cartilage and vertebral cartilage.
- the cell therapeutic agent of the present invention may be used for treating bone defect, tendon-ligament defect, adipose tissue defect, cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma, arthritis, cartilage deficiency or congenital organ softening.
- the cell therapy agent or the composition for tissue regeneration of the present invention may be administered directly into a joint or joint cavity, and in this case, it may be administered in the form of an injection.
- the stem cells derived from the decidua adjacent chorionic space of the present invention are stem cells with excellent differentiation ability that can be differentiated into cartilage, bone, fat, etc., they can be administered to joints, cartilage, tendons, or ligaments by administering to various lesions, such as lesions of articular cartilage. can be treated or prevented.
- intra-articular administration of the stem cells derived from the decidua adjacent chorionic space of the present invention can regenerate the cartilage surface in a soft and almost free state, and protect and regenerate cartilage so that arthritis does not progress.
- composition for cell therapy or tissue regeneration of the present invention may be formulated in the form of an injection for direct administration of stem cells derived from the decidua adjacent chorionic space, and in this case, may further include a hydrogel suitable for injection.
- the hydrogel that can be used in the cell therapy of the present invention may include, without limitation, a hydrogel suitable for injection, known in the art, small intestine submucosal tissue, hyaluronic acid, carboxymethylcellulose (CMC, carboxymethylcellulose), Alginate, chitosan, polyacrylamide, poly (N-isopropylacrylamide) (poly (N-isopropylacrylamide)), ⁇ glycerophosphate ( ⁇ pluronic (Poly (ethylene oxide)) poly(propylene oxide) poly(ethyleneoxide), Pluronic), fibrin, polyethylene oxide (PEO) and at least one selected from the group consisting of a mixture of carboxymethyl cellulose (CMC) and polyethyleneimine (PEI), preferred of the present invention
- hyaluronic acid more specifically sodium hyaluronate, was used by mixing with stem cells derived from the decidua adjacent interchorionic space.
- the preferred dosage of the cell therapy agent of the present invention varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. Administration may be administered once a day or may be administered in several divided doses, and the dosage is not intended to limit the scope of the present invention in any way.
- the subject subject to treatment and administration of the decidual-proximal interchorionic space-derived stem cell or cell therapeutic agent containing the same of the present invention is damaged or deleted at least one tissue selected from the group consisting of fat, cartilage, bone, nerve, ligament, and tendon. It may be an individual in need or foreseeable need for tissue regeneration.
- the cell therapy agent of the present invention when administered to an individual, it can be used without limitation as a formulation known in the art as long as it can achieve the effect of the stem cell therapy and effectively induce tissue regeneration in the individual, preferably in the form of an injection. It can be formulated and administered.
- the stem cell derived from the decidua adjacent chorionic space according to the present invention has excellent proliferation and differentiation ability and thus has excellent tissue regeneration effect, and the content overlapping with the content described in the cell therapy for tissue regeneration is omitted to avoid the complexity of the description of the specification.
- Example 1 Separation of the interchorionic cavity adjacent to the placental decidua and obtaining stem cells
- the trophoblast layer adjacent to the decidua was defined as the trophoblast layer adjacent to the decidua among the total trophoblasts, and when the entire trophoblast was divided into thirds, it was defined as the trophoblast layer from the area adjacent to the decidua to the 2/3 point.
- the trophoblast layer adjacent to the decidua is again separated into villi and intervillous, and then the villi are removed. It was washed more than 5 times to In order to distinguish the obtained interchorionic space tissue separated from the trophoblast layer adjacent to the decidua from the entire intervillous space (IVS) tissue, it was defined as 'the decidua adjacent interchorionic space (CT-V2)'.
- CT-V2 decidua adjacent interchorionic space
- CT-V2 tissue of the interchorionic space
- a-MEM medium supplemented with 0.2% collagenase was added, and the mixture was reacted for 2 hours using a stirrer at 37 ° C.
- Cells derived from The obtained CT-V2 derived cells were filtered through a 100 ⁇ m mesh to remove undecomposed tissue, and a-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25° C. and 1500 rpm for 4 minutes.
- a-MEM medium containing no growth factors and containing fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at 37° C. under 5% CO 2 conditions.
- cells attached to the bottom of the culture vessel were selected to obtain stem cells derived from CT-V2.
- Comparative Example 1 Obtaining stem cells derived from other tissues
- the whole placental tissue was cut into about 5*5*3cm and washed with PBS to remove blood and blood cells from the placental tissue.
- A-MEM medium supplemented with 0.2% collagenase was added to the washed placental tissue and reacted using a stirrer at 37° C. to obtain placental cells.
- the obtained placental cells were filtered through a 100 ⁇ m mesh to remove undecomposed tissue, and a-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25° C. and 1000 rpm for 4 minutes.
- a-MEM medium containing no growth factors and containing fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at 37° C. under 5% CO 2 conditions. Cells attached to the bottom of the culture vessel were selected from the culture to obtain stem cells derived from whole placenta (Pla).
- the entire trophoblast layer with a size of about 5*5*3cm was obtained and transferred to a 150mm dish, and washed more than 5 times with PBS to remove blood and blood cells.
- the entire trophoblast was separated again into villi and intervillous, and then the separated intervillous was used 5 times to remove the remaining blood and blood cells using PBS. It was washed over.
- the remaining tissue part except for the decidual adjacent interchorionic space (CT-V2) described in Example 1 was defined as CT-V1.
- a-MEM medium supplemented with 0.2% collagenase was added, and the cells derived from each tissue were reacted for 2 hours at 37°C using a stirrer. obtained.
- the obtained cells were filtered through a 100 ⁇ m mesh to remove undecomposed tissue, and a-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25° C. and 1500 rpm for 4 minutes. After removing the supernatant, a-MEM medium containing no growth factors and containing fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at 37° C. under 5% CO 2 conditions.
- Example 2 Decidual adjacent interchorionic space (CT-V2) derived stem cell culture and cell morphology confirmation
- the a-MEM medium containing no growth factors, fetal bovine serum and antibiotics was added and cultured while replacing every 2-3 days.
- the stem cells have grown more than 80%, the stem cells are separated from the culture vessel by treating the triple (TryPLE), the separated stem cells are diluted at a ratio of 1/5, and then passaged by culturing in another culture vessel. Incubation was performed. Cell morphology after culture was observed under a microscope.
- CT-V2-derived stem cells As shown in FIG. 2 , it was confirmed that the CT-V2-derived stem cells according to the present invention exhibited morphological characteristics of fibroblasts and specifically maintained only single cells. On the other hand, whole placenta (Pla) and other detailed tissue-derived stem cells showed fibroblast-like morphological characteristics, but it was confirmed that a number of different types of cells were mixed.
- CT-V2-derived stem cells specifically maintained only single cells before and after subculture, but stem cells derived from stem cells derived from the whole placenta and other detailed tissues were mixed with cells of different types. Confirmed.
- Example 3 Analysis of colony formation performance of stem cells derived from the decidua adjacent interchorionic space (CT-V2)
- the colony-forming ability of the CT-V2-derived stem cells obtained in Example 1 was confirmed. More specifically, the first subculture of the CT-V2-derived stem cells obtained in Example 1 was performed by the method of Example 2, and 5 X 10 3 each in a 100 mm dish at the end of the subculture. After inoculation (seeding), it was cultured in a-MEM medium supplemented with fetal bovine serum and antibiotics without growth factors for 14 days. Thereafter, the number of colonies formed in each stem cell was counted. In addition, using the whole placenta obtained in Comparative Example 1 and stem cells derived from other placental detailed tissues, population doubling time and colony forming ability were measured in the same manner. In the case of colony-forming ability, the result value of whole placental-derived stem cells was converted into 100%. The results are shown in FIGS. 3 and 4 .
- CT-V2-derived stem cells are derived from the entire placenta (Pla), the entire interchorionic cavity (IVS), and the chorionic plate adjacent interchorionic cavity (CT-V1) excluding the decidual adjacent interchorionic space from the entire interchorionic cavity. It was confirmed that the colony forming ability was significantly superior to that of stem cells.
- CT-V2-derived stem cells exhibited more than double the colony-forming ability compared to CT-V1-derived stem cells, and that they were very excellent in colony-forming ability compared to whole villi and other detailed tissue-derived stem cells.
- the population doubling time was maintained similarly until P2, but after P2, the CT-V2-derived stem cells of the present invention exhibited the highest cumulative population doubling level, indicating the lowest population doubling time. .
- the stem cells derived from the villi (CT-V2) adjacent to the chorionic plate were stem cells with excellent proliferative capacity.
- Example 4 Analysis of surface markers of stem cells derived from decidual adjacent interchorionic space (CT-V2)
- Example 1 the interchorionic cavity adjacent to the decidua was obtained, washed with PBS, triple-treated, and then stem cells were collected and centrifuged at 1200 rpm for 5 minutes. After removing the supernatant, the stem cells were washed with PBS, and centrifuged at 1200 rpm for 5 minutes. After removing the supernatant, the stem cells are suspended in PBS, passed through a 40 ⁇ m cell strainer, and the cells and BD Pharmingen Stain Buffer + EDTA are prepared and mixed. Falcon® round-bottom 96-well plate (Cat. 353910) 100 ⁇ l was dispensed into each well (100,000 pieces/well).
- BD Pharmingen Stain Buffer + EDTA was added to each well, centrifuged at 1200 rpm for 5 minutes, the supernatant was removed, and the washing process was carefully repeated twice using PBS. The supernatant was removed, and 150 ⁇ l of BD Pharmingen Stain Buffer + EDTA was dispensed into each well. At least 10,000 cells per well were analyzed for surface markers using a flow cytometer (FACS). In addition, the surface markers of the whole placenta obtained in Comparative Example 1 and other placental detail-derived stem cells were analyzed in the same manner. The results are shown in Tables 1 and 2.
- the expression level of the surface marker is less than 5%, it is classified as a negative marker, and if it is 5% or more, it is classified as low if it is 5% or more and less than 30% among positive markers, middle if it is 30 to less than 70%, and high if it is 70% or more according to the expression level. According to these criteria, the expression values of the markers in Tables 1 and 2 were summarized, and the expression of the markers was shown in Tables 3 and 4.
- CT-V2 of the present invention showed high expression of CD49b and CD200 markers, unlike whole placental, IVS, and CT-V2-derived stem cells, and unlike other stem cells, CD24 and CD227 It was confirmed that this negative surface marker expression pattern was shown. Through this difference in expression, it was confirmed that the CT-V2-derived stem cells are new stem cells that exhibit completely different CD marker expression characteristics from other placental detailed tissue-derived stem cells.
- the above results show that stem cells derived from the whole villi are in a state in which stem cells showing various CD marker expression patterns are mixed. It can be confirmed that one stem cell can be obtained.
- Example 5 Confirmation of differentiation ability into adipocytes of stem cells CT-V2 derived from the interchorionic space adjacent to the decidua
- the stem cells were subjected to a known adipocyte differentiation induction medium 1 (10% FBS, 1% Anti-biotics, 1 ⁇ M dexamethasone, DMEM medium with 20 ⁇ M indomethacin, 10 ⁇ M insulin, 50 ⁇ M 3-isobutyl-1-methylxanthine (IBMX)) and adipocyte differentiation induction medium 2 (DMEM with 10% FBS, 1% Anti-biotics, 10 ⁇ M insulin) medium) was alternately added for 3 to 4 days and cultured for 3 weeks to induce differentiation into adipocytes.
- adipocyte differentiation induction medium 1 10% FBS, 1% Anti-biotics, 1 ⁇ M dexamethasone, DMEM medium with 20 ⁇ M indomethacin, 10 ⁇ M insulin, 50 ⁇ M 3-isobutyl-1-methylxanthine (IBMX)
- adipocyte differentiation induction medium 2 DMEM with 10% FBS, 1% Anti-biotics, 10 ⁇ M
- CT-V2-derived stem cells according to the present invention had superior adipocyte differentiation ability compared to IVS and CT-V1-derived stem cells.
- Example 6 Confirmation of chondrocyte differentiation ability of stem cells derived from decidua adjacent interchorionic space (CT-V2)
- the stem cells were subjected to a known chondrocyte differentiation induction medium (0.1 ⁇ M dexamethasone, 50 ⁇ g). /ml ascorbic acid, 40 ⁇ g/ml L-proline, 10ng/ml TGF- ⁇ 500ng/ml BMP-6, 50mg/ml ITS premix (DMEM medium containing premix) for 3 weeks to induce differentiation into chondrocytes .
- a known chondrocyte differentiation induction medium 0.1 ⁇ M dexamethasone, 50 ⁇ g.
- /ml ascorbic acid 40 ⁇ g/ml L-proline
- 10ng/ml TGF- ⁇ 500ng/ml BMP-6 50mg/ml ITS premix
- DMEM medium containing premix 50mg/ml ITS premix
- CT-V2-derived stem cells according to the present invention had superior chondrocyte differentiation ability compared to IVS and CT-V1-derived stem cells.
- Example 7 Confirmation of differentiation ability of stem cells derived from decidua adjacent interchorionic space (CV-V2) into osteocytes
- the stem cells were subjected to a known osteocytic differentiation induction medium (10% FBS, 1% Anti-biotics, 100 ⁇ M dexamethasone, 50 mM ascorbic acid-2-phosphate, 10 ⁇ M ⁇ -glycrophosphate, and DMEM medium containing 250 ⁇ M ascorbic acid) were cultured for 4 weeks to induce differentiation into osteocytes.
- a known osteocytic differentiation induction medium (10% FBS, 1% Anti-biotics, 100 ⁇ M dexamethasone, 50 mM ascorbic acid-2-phosphate, 10 ⁇ M ⁇ -glycrophosphate, and DMEM medium containing 250 ⁇ M ascorbic acid
- the degree of differentiation into furnaces was analyzed.
- 0.6M HCl was treated for 24 hours to measure intracellular calcium at 4 weeks after the differentiation ability of the whole placenta and other placental subtissue-derived stem cells obtained in Comparative Example 1 into osteocytes in the same manner.
- absorbance was measured at 565 nm using the o-cresolphthalein complexone method (Pointe Scientific, Canton, MI, USA), and calcium concentration was standardized and quantified. The results are shown in FIG. 7 .
- the stem cells derived from the decidual interchorionic space (CT-V2) according to the present invention have superior osteocytic differentiation ability that can be differentiated into osteocytes compared to the stem cells isolated from IVS and CT-V1. confirmed that there is.
- Example 8 Verification of the cell therapeutic effect of stem cells derived from the decidua adjacent chorionic space (CT-V2) (cartilage defect animal model)
- Example 1 In order to verify the effect of stem cells derived from the decidual adjacent villi (CT-V2) obtained in Example 1 as a cell therapeutic agent in an animal model of cartilage defect, the following experiment was performed. More specifically, in order to produce an 18-week-old rat articular cartilage damage animal model, a healthy rat was selected and anesthetized with appropriate amounts of ketamine and rumpun according to body weight, and then it was confirmed that the rat was sufficiently under general anesthesia, After shaving the knee joint of both lower extremities, it was fixed with a band-aid while maintaining the posture.
- CTL2 decidual adjacent villi
- the defect site was found to be irregular with a recessed surface compared to the surrounding normal cartilage tissue. , cracks or cracks were observed on the surface of the defect site, and close union with surrounding normal cartilage was not observed.
- the newly formed tissue and normal articular cartilage were well combined, resulting in a more mature and complete cell arrangement in the superficial and deep layers. A morphology similar to that of normal cartilage was also observed.
- CT-V2 stem cells appear to play a role in regenerating damaged cartilage.
- the conventional stem cells derived from the whole placenta are mixed with stem cells derived from detailed tissues having various characteristics, so the ability to differentiate into different cells does not appear uniformly, whereas the placenta according to the present invention is not uniformly displayed.
- stem cells derived from the decidua adjacent interchorionic space (CT-V2) a detailed tissue of could
- CT-V2-derived stem cells according to the present invention showed consistent aspects in the characteristics of growth, proliferation, morphology and differentiation than other placental tissue-derived stem cells, and showed the best stem cell characteristics. Therefore, it was confirmed that, when the CT-V2-derived stem cells are used, the differentiation efficiency into a target cell can be improved, and it can be usefully used as a cell therapy agent in various diseases.
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Abstract
The present invention relates to stem cells derived from intervillous space tissue, preferably an intervillous space adjacent to the decidua, and a cellular therapeutic agent comprising same. The stem cells derived from an intervillous space adjacent to the decidua, according to the present invention, exhibit uniform growth characteristics and excellent proliferation characteristics and have a significantly excellent ability to differentiate into cartilage, bone and fat, as compared to other placental tissue-derived stem cells, and thus can be effectively used in various tissue regeneration treatments needed for the regeneration of cartilage, bone and fat, particularly in cartilage regeneration and osteoarthritis treatments.
Description
본 발명은 융모간강 조직, 바람직하게는 탈락막 인접 융모간강으로부터 유래된 줄기세포 및 이를 포함하는 세포치료제에 관한 것이다.The present invention relates to stem cells derived from interchorionic cavity tissue, preferably from the interchorionic space adjacent to the decidua, and a cell therapy product containing the same.
줄기세포 (stem cell) 란, 자기 복제 능력을 가지면서 두 개 이상의 세포로 분화하는 능력을 갖는 세포를 말하며, 만능 줄기세포 (totipotent stem cell), 전능성 (전분화능) 줄기세포 (pluripotent stem cell), 다능성 (다분화능) 줄기세포 (multipotent stem cell)로 분류할 수 있다. 만능 줄기세포 (totipotent stem cell) 는 하나의 완전한 개체로 발생해 나갈 수 있는 만능의 성질을 가진 세포로 난자와 정자의 수정 이후 8세포기까지의 세포가 이러한 성질을 가지며 이 세포를 분리하여 자궁에 이식하면 하나의 완전한 개체로 발생해 나갈 수 있다. 전분화능 줄기세포 (pluripotent stem cells) 는 외배엽, 중배엽, 내배엽 유래의 다양한 세포와 조직으로 발생할 수 있는 세포로, 수정 4-5일 후 나타나는 배반포 (blastocyst) 의 안쪽에 위치한 내세포괴 (inner cell mass)에서 유래하며 이를 배아 줄기세포라 하며 다양한 다른 조직세포로 분화되지만 새로운 생명체를 형성하지는 못한다. 다분화능 줄기세포 (multipotent stem cells)는 이 세포가 포함되어 있는 조직 및 기관에 특이적인 세포로만 분화할 수 있는 줄기세포이다. 상기와 같은 다분화능 줄기세포의 소스로는 성체 골수, 피부, 혈관, 근육 등이 알려져 있고, 조직공학, 유전자 치료분야 및 세포치료제 분야 등에도 이러한 줄기세포들이 급속도로 적용되고 있으며, 이외에도 여러 조직으로부터 줄기세포를 얻고, 이에 대한 다양한 응용이 절실히 요구되고 있는 상황이다.Stem cell refers to a cell that has the ability to differentiate into two or more cells while having the ability to self-replicate, totipotent stem cell, pluripotent stem cell, It can be classified as a multipotent (multipotent) stem cell. A totipotent stem cell is a cell with pluripotent properties that can develop into a complete individual. Cells up to the 8th cell stage after fertilization of an egg and sperm have these properties. Transplantation can develop into a complete individual. Pluripotent stem cells are cells that can develop into various cells and tissues derived from ectoderm, mesoderm, and endoderm. The inner cell mass is located inside the blastocyst that appears 4-5 days after fertilization. These are called embryonic stem cells, and they differentiate into various other tissue cells, but do not form new life forms. Multipotent stem cells are stem cells that can differentiate only into cells specific to the tissues and organs that contain these cells. Adult bone marrow, skin, blood vessels, muscle, etc. are known as sources of pluripotent stem cells as described above, and these stem cells are rapidly being applied to tissue engineering, gene therapy, and cell therapy fields. It is a situation in which stem cells are obtained and various applications thereof are urgently required.
태반은 여성이 임신했을 때, 자궁벽에서 발생하는 장기로 혈관조직이 풍부한 원반형 형태를 보이며, 태아의 영양섭취와 호흡, 배설 등이 모두 태반을 통해 이루어진다. 일생의 건강이 태아시기에 결정된다는 과학적 연구 결과들이 보고되면서, 임신 기간 중의 태반의 중요성이 부각되고 있으며, 이에 관련된 여러 가지 물질들의 상호관계를 규명하고자 많은 노력이 시도되고 있다. 태반은, 주수별, 위치별 다양한 형태의 세포들로 구성되어 있는데, 아직은 그 활용범위가 극히 제한적으로 이루어지고 있다. The placenta is an organ that develops from the uterine wall when a woman is pregnant, and has a disk-like shape rich in vascular tissues. As scientific research results that lifelong health is determined during the fetal period are reported, the importance of the placenta during pregnancy is being emphasized, and many efforts are being made to clarify the interrelationship between various substances related to it. The placenta is composed of various types of cells by age and location, but its application is still very limited.
최근에는, 태반조직으로부터 분리된 줄기세포에 관한 연구들도 진행되고 있다. 종래의 태반 조직 유래 줄기세포는 출산 시에 얻을 수 있는 만삭 태반의 일부 조직으로부터 유래된 줄기세포가 대부분이었다. 그러나 태반은 자궁에 붙어있는 기저막, 태반의 중심조직으로 모체와 태아 혈액간에 물질 교환을 매개하는 융모조직, 태반의 안쪽으로 태반을 덮고 있으면서 태아와 양막을 싸고있는 융모막 등 다양한 조직으로 이루어져 있으며, 아직까지 이들 세부 조직에 대한 연구는 널리 알려지지 않았다. 한편 대한민국 특허등록 제818214호에는 NAC(N-acrtyl-L-cysteine) 함유 배지를 이용하여 양막 또는 탈락막으로부터 줄기세포를 분리하는 방법이 개시되어 있고, 대한민국 특허등록 제871984호에는 bFGF(Basic Fibroblast Growth Factor) 함유 배지를 이용하여 양막, 장막, 기저 탈락막 및 태반 조직으로부터 유래된 줄기세포의 다분화능에 관해 개시하고 있다. Recently, studies on stem cells isolated from placental tissue are also being conducted. Most of the conventional placental tissue-derived stem cells are stem cells derived from some tissues of a term placenta obtained during childbirth. However, the placenta is composed of various tissues such as the basement membrane attached to the uterus, the chorionic tissue that mediates the exchange of substances between mother and fetal blood as the central tissue of the placenta, and the chorionic membrane that covers the placenta inside the placenta and surrounds the fetus and the amniotic membrane. Until now, studies on these sub-organisms were not widely known. Meanwhile, Korean Patent Registration No. 818214 discloses a method for isolating stem cells from amniotic membrane or decidua using NAC (N-acrtyl-L-cysteine)-containing medium, and Korean Patent Registration No. 871984 discloses bFGF (Basic Fibroblast). Growth Factor)-containing medium is disclosed for the pluripotency of stem cells derived from amniotic membrane, serous membrane, basal decidua and placental tissues.
그러나 종래 해부학적으로 알려진 태반 구조 구별방법에 구애되지 않고 태반을 세분화하고, 세분화된 부위로부터 얻어지는 줄기세포 및 이의 효과에 대해서는 아직까지 널리 보고되지 않았다. However, stem cells obtained from subdivided placenta and subdivided sites and their effects have not yet been widely reported, regardless of conventional anatomically known methods for distinguishing placenta structures.
본 발명자들은 태반의 세부 부위를 새롭게 정의하고 이에 따른 줄기세포의 특성을 연구하던 중, 탈락막 인접 융모간강(CT-V2)을 수득하였고 이로부터 줄기세포를 분리하였다. 분리된 탈락막 인접 융모간강 유래 줄기세포는 전체 태반(Whole placenta), 전체 융모간강(intervillous space, IVS), 전체 융모간강 중 탈락막 인접 융모간강 부위를 제외한 융모막판 인접 융모간강 (CT-V1) 유래 줄기세포들과 CD 마커 발현 패턴이 상이하고, 집락 형성능, 증식력, 연골, 골 및 지방으로의 분화능이 모두 현저히 상이함을 확인하고 본 발명을 완성하였다.While the present inventors newly defined the detailed region of the placenta and studied the stem cell characteristics accordingly, the decidual adjacent interchorionic cavity (CT-V2) was obtained and stem cells were isolated therefrom. The isolated decidual-proximal interchorionic space-derived stem cells were obtained from the whole placenta, the entire intervillous space (IVS), and the chorionic plate-adjacent interchorionic space (CT-V1) The present invention was completed by confirming that the derived stem cells and CD marker expression patterns were different, and the colony forming ability, proliferative ability, and differentiation ability into cartilage, bone and fat were all significantly different.
따라서 본 발명의 목적은 탈락막 인접 융모간강 유래 줄기세포 및 이를 이용한 세포치료제, 조직 재생용 조성물, 이들의 분리 방법을 제공하는 것이다.Accordingly, it is an object of the present invention to provide stem cells derived from the interchorionic cavity adjacent to the decidua, a cell therapy agent using the same, a composition for tissue regeneration, and a method for separating them.
상기 목적을 달성하기 위하여, 본 발명은 탈락막 인접 융모간강 유래 줄기세포로, 상기 탈락막 인접 융모간강은 전체 영양막 층 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인, 탈락막 인접 융모간강 유래 줄기세포를 제공한다. In order to achieve the above object, the present invention is a stem cell derived from the decidua adjacent interchorionic space, wherein the decidual adjacent interchorionic space is a trophoblast from the decidua adjacent site to the 2/3 point of the entire trophoblast layer, the decidua adjacent villi Provided is a liver-derived stem cell.
또한 본 발명은 1) 전체 영양막층 중 탈락막 인접 부위에서부터 2/3 지점까지인 영양막층 부위를 절단하여 탈락막 인접 영양막층을 얻는 단계; 2) 상기 탈락막 인접 영양막층에서 융모를 제거하여 탈락막 인접 융모간강을 얻는 단계; 및 3) 상기 탈락막 인접 융모간강에서 줄기세포를 수득하는 단계; 를 포함하는 탈락막 인접 융모간강 유래 줄기세포의 분리 수득 방법을 제공한다. In addition, the present invention comprises the steps of: 1) cutting the trophoblast layer from the adjacent decidua to 2/3 of the entire trophoblast layer to obtain a trophoblast adjacent to the decidua; 2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; It provides a method for separating and obtaining stem cells derived from the interchorionic space adjacent to the decidua, comprising a.
또한 본 발명은 상기 줄기세포를 유효성분으로 포함하는 조직 재생용 세포치료제를 제공한다. In addition, the present invention provides a cell therapy agent for tissue regeneration comprising the stem cells as an active ingredient.
또한 본 발명은 상기 줄기세포를 유효성분으로 포함하는 조직 재생용 조성물을 제공한다. The present invention also provides a composition for tissue regeneration comprising the stem cells as an active ingredient.
또한 본 발명은 전체 영양막 층 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인 탈락막 인접 융모간강 유래 줄기세포의 조직 재생 용도를 제공한다. In addition, the present invention provides a tissue regeneration use of stem cells derived from the decidua adjacent to the trophoblast, which is the trophoblast of the region from the region adjacent to the decidua to the 2/3 point of the entire trophoblast layer.
또한 본 발명은 전체 영양막 층 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인 탈락막 인접 융모간강 유래 줄기세포의 조직 재생용 세포 치료제 용도를 제공한다. In addition, the present invention provides the use of a cell therapeutic for tissue regeneration of stem cells derived from the decidua adjacent to the trophoblast, which is the trophoblast in the region from the region adjacent to the decidua to the 2/3 point of the entire trophoblast layer.
또한 본 발명은 전체 영양막 층 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인 탈락막 인접 융모간강 유래 줄기세포를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 조직 재생방법을 제공한다. In addition, the present invention provides a tissue regeneration method, comprising administering to an individual in need thereof, stem cells derived from the decidua adjacent to the trophoblast, which are the trophoblasts of the region from the region adjacent to the decidua to the 2/3 point of the entire trophoblast layer. .
본 발명에 따른 탈락막 인접 융모간강 유래 줄기세포는 다른 태반 조직 유래 줄기세포와 비교하여 균질한 성장 특성, 우수한 증식 특성을 나타내고, 연골, 골 및 지방으로의 분화능이 현저히 우수하므로 연골, 골 및 지방의 재생이 필요한 다양한 조직 재생 치료, 특히 연골 재생 및 골 관절염 치료에 효과적으로 활용될 수 있다.The decidua-proximal interchorionic space-derived stem cells according to the present invention exhibit homogeneous growth characteristics and excellent proliferation characteristics compared to other placental tissue-derived stem cells, and have remarkably excellent ability to differentiate into cartilage, bone and fat. It can be effectively used for various tissue regeneration treatments that require the regeneration of bone, especially cartilage regeneration and osteoarthritis treatment.
도 1은 전체 태반(Whole placenta), 전체 융모간강(intervillous space, IVS), 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 의 모식도이다.1 is a schematic diagram of the whole placenta, the entire intervillous space (IVS), the chorionic plate adjacent interchorionic space (CT-V1), and the decidual adjacent interchorionic space (CT-V2).
도 2는 전체 태반(Whole placenta, Pla), 전체 융모간강(intervillous space, IVS), 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포를 배양한 후 세포 형태를 현미경으로 관찰한 결과를 나타낸 도이다.Figure 2 is after culturing stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2); It is a diagram showing the result of observing the cell morphology under a microscope.
도 3은 전체 태반(Whole placenta, Pla), 전체 융모간강(intervillous space, IVS), 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 집락형성능을 확인한 결과를 나타낸 도이다(*P>0.05, ***P>0.001).Figure 3 shows the colony forming performance of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1), and decidual adjacent interchorionic space (CT-V2); It is a diagram showing the confirmed results (*P>0.05, ***P>0.001).
도 4은 전체 태반(Whole placenta, Pla), 전체 융모간강(intervillous space, IVS), 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 집단 배가 시간을 비교한 결과를 나타낸 도이다.4 shows the population doubling time of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1), and decidual adjacent interchorionic space (CT-V2); It is a diagram showing the results of comparison.
도 5은 전체 태반(Whole placenta, Pla), 전체 융모간강(intervillous space, IVS), 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 지방세포 분화능을 확인한 결과를 나타낸 도이다(*P>0.05, **P>0.01).Figure 5 shows adipocyte differentiation ability of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1), and decidual adjacent interchorionic space (CT-V2); is a diagram showing the results of confirming (*P>0.05, **P>0.01).
도 6은 전체 태반(Whole placenta, Pla), 전체 융모간강(intervillous space, IVS), 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 연골세포 분화능을 확인한 결과를 나타낸 도이다(**P>0.01, ***P>0.001).6 is a chondrocyte differentiation ability of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2); is a diagram showing the results of confirming (**P>0.01, ***P>0.001).
도 7은 전체 태반(Whole placenta, Pla), 전체 융모간강(intervillous space, IVS), 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 골세포 분화능을 확인한 결과를 나타낸 도이다(*P>0.05, ***P>0.001).7 shows the osteocytic differentiation potential of stem cells derived from whole placenta (Pla), whole intervillous space (IVS), chorionic plate adjacent interchorionic space (CT-V1), and decidual adjacent interchorionic space (CT-V2); is a diagram showing the results of confirming (*P>0.05, ***P>0.001).
도 8은 래트 연골 결손 동물 모델에 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포를 히알루론산(HA)과 혼합 투여 후, 연골 재생 효과를 육안으로 평가한 결과를 나타낸 도이다. 8 is a view showing the cartilage regeneration effect after mixed administration of stem cells derived from the interchorionic space adjacent to the chorionic plate (CT-V1) and the decidua adjacent interchorionic space (CT-V2) with hyaluronic acid (HA) to the rat cartilage defect animal model. It is a figure which shows the evaluation result.
도 9는 래트 연골 결손 동물 모델에 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포와 히알루론산을 혼합 투여 후, 재생 효과를 ICRS macroscopic score 를 이용하여 평가한 결과를 나타낸 도이다(*P>0.01, **P>0.001, ***P>0.001). Figure 9 is a rat cartilage defect animal model after chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2) derived stem cells and hyaluronic acid after mixed administration, the regeneration effect using the ICRS macroscopic score It is a diagram showing the evaluation results (*P>0.01, **P>0.001, ***P>0.001).
도 10은 래트 연골 결손 동물 모델에 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포와 히알루론산을 혼합 투여 후, H&E, Safranin O 및 type II collagen 염색을 통해 재생 효과를 확인한 결과를 나타낸 도이다. Figure 10 is after mixed administration of stem cells derived from the chorionic interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2) and hyaluronic acid to the rat cartilage defect animal model, H&E, Safranin O and type II collagen staining It is a diagram showing the results of confirming the regeneration effect through
도 11은 래트 연골 결손 동물 모델에 융모막판 인접 융모간강(CT-V1) 및 탈락막 인접 융모간강(CT-V2) 유래 줄기세포와 히알루론산을 혼합 투여 후, 재생 효과를 Modified O'driscoll score 를 통해 확인한 결과를 나타낸 도이다(*P>0.01, **P>0.001, ***P>0.001). Figure 11 is after mixed administration of stem cells and hyaluronic acid derived from the chorionic plate adjacent interchorionic space (CT-V1) and decidual adjacent interchorionic space (CT-V2) to the rat cartilage defect animal model, Modified O'driscoll score for the regeneration effect It is a diagram showing the results confirmed through (*P>0.01, **P>0.001, ***P>0.001).
본 발명은 탈락막 인접 융모간강 (intervillous space) 유래 줄기세포 및 이를 포함하는 조직재생용 세포 치료제, 조직 재생용 조성물을 제공한다. The present invention provides a stem cell derived from the decidual space adjacent to the decidua, a cell therapeutic agent for tissue regeneration comprising the same, and a composition for tissue regeneration.
이하, 본 발명에 대해 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 탈락막 인접 융모간강 유래 줄기세포로, 상기 탈락막 인접 융모간강은 전체 영양막 층 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인, 탈락막 인접 융모간강 유래 줄기세포를 제공한다. The present invention provides stem cells derived from the decidua adjacent interchorionic space, wherein the decidual adjacent interchorionic space is a trophoblast of the entire trophoblast adjacent to the decidua to 2/3 of the entire trophoblast layer. .
본 발명에서 "줄기세포"란, 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 의미한다. 줄기세포는 분화능에 따라, 만능 줄기세포(totipotent stem cell), 전분화능 줄기세포(pluripotent stem cells), 다분화능(다능성) 줄기세포(multpotent stem cells)로 분류할 수 있다. As used herein, the term "stem cell" refers to a cell having the ability to differentiate into two or more different types of cells while having the ability to self-replicate. Stem cells may be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells according to their differentiation capacity.
본 발명에서 "태반(placenta)"이란, 임신 중에 태아를 위해 만들어지는 생체 내 조직을 의미하는데, 무게 500-600 g, 지름 15-20 cm, 두께 2-3 cm 정도의 원반형태이다. 태반의 한쪽은 모체와 닿아 있고 다른 한쪽은 태아와 맞닿아 있으며, 그 사이에서 모체의 혈액과 태아의 혈관 사이에 영양분 및 산소의 전달이 이루어지게 된다. 태반은 크게 양막, 융모막, 탈락막의 3층으로 구분할 수 있고, 보다 상세하게는 양막 상피, 양막, 융모막, 영양막, 탈락막으로 구분할 수 있다. In the present invention, "placenta" means an in vivo tissue made for a fetus during pregnancy, and is in the form of a disc having a weight of 500-600 g, a diameter of 15-20 cm, and a thickness of about 2-3 cm. One side of the placenta is in contact with the mother and the other side is in contact with the fetus, between which nutrients and oxygen are transferred between the blood vessels of the mother and the fetus. The placenta can be largely divided into three layers of amniotic membrane, chorion, and decidua, and more specifically, can be divided into amniotic epithelium, amniotic membrane, chorion, trophoblast, and decidua.
본 발명에서는 상기 태반 중 영양막층(Chorion) 에 존재하는 융모간강 영역 (intervillous space, IVS) 을 보다 세부적으로 구분하여, 탈락막 인접 영양막 층의 융모간강을 수득하고 이로부터 줄기세포를 얻는 것을 특징으로 한다. '탈락막 인접 영양막층' 은 전체 영양막 중 탈락막 쪽에 위치한 영양막층으로, 전체 영양막층을 3등분 하였을 때, 탈락막 인접 부위에서부터 2/3 지점까지의 연속적인 부위로 정의된다. 상기 탈락막 인접 영양막층에서 융모(villi)가 제외된 부위를 '탈락막 인접 융모간강' 으로 정의하였고, 본 발명에서는 'CT-V2' 로 명명하였다. In the present invention, the intervillous space (IVS) present in the trophoblast layer (Chorion) of the placenta is divided in more detail to obtain the interchorionic space of the trophoblast layer adjacent to the decidua, and stem cells are obtained therefrom. do. The 'trophic layer adjacent to the decidua' is a trophic layer located on the decidua side of the total trophoblast. When the entire trophoblast layer is divided into three, it is defined as a continuous region from the adjacent decidua to 2/3 points. The region in which villi were excluded from the trophoblast layer adjacent to the decidua was defined as the 'intervilli space adjacent to the decidua,' and in the present invention, it was designated as 'CT-V2'.
한편, 전체 영양막층 내 융모간강 부위 중 상기 탈락막 인접 융모간강 부위를 제외한 나머지 부분은 탈락막 반대쪽의 융모막판 (chorionic plate) 에 인접해 있는 융모간강이며, 본 발명에서는 이를 '융모막판 인접 융모간강'으로 정의하고, 'CT-V1'으로 명명하였다. 상기 융모막판 인접 융모간강 부위는 전체 영양막층을 3등분 하였을 때, 융모막판 인접부위에서부터 1/3 지점까지의 연속적인 부위인 영양막 층에서 융모를 제외한 부위로 정의된다. On the other hand, in the entire trophoblast layer, the remaining part except for the interchorionic space adjacent to the decidua is the interchorionic space adjacent to the chorionic plate on the opposite side of the decidua, and in the present invention, this ' and named 'CT-V1'. The chorionic plate adjacent interchorionic cavity region is defined as a region excluding the villi in the trophoblast layer, which is a continuous region from the chorionic plate adjacent region to 1/3 point when the entire trophoblast layer is divided into thirds.
본 발명에서는 탈락막 인접 융모간강(CT-V2) 유래 줄기세포가 전체 태반, 전체 융모간강 및 융모막판 인접 융모간강 유래 줄기세포와 비교하여 CD 마커 발현 패턴이 상이하고, 균질한 성장 특성, 우수한 증식 특성을 나타내며, 연골, 골 및 지방으로의 분화능이 현저히 우수함을 확인하였다. In the present invention, stem cells derived from the decidual interchorionic space (CT-V2) have a different CD marker expression pattern compared to stem cells derived from the whole placenta, the entire interchorionic space and the chorionic plate adjacent interchorionic space, and have homogeneous growth characteristics and excellent proliferation. It was confirmed that the ability to differentiate into cartilage, bone and fat was remarkably excellent.
보다 구체적으로 본 발명의 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 CD24, CD104, CD227, Disialogangolioside GD2 또는 SSEA-4 에 대하여 음성의 표면인자 발현 특성을 갖거나; 또는 CD49b, CD74 또는 CD200에 대하여 양성의 표면인자 발현 특성을 갖는 것을 특징으로 할 수 있고 바람직하게는 CD24, CD104, CD227, Disialogangolioside GD2 및 SSEA-4로 이루어진 군에서 선택된 1종 이상, 2종 이상, 3종 이상, 4종 이상, 5종 모두의 표면인자가 모두 음성이고/이거나 CD49b, CD74 및 CD200로 이루어진 군에서 선택된 1종 이상, 2종 이상, 3종 모두의 표면인자가 모두 양성일 수 있다. More specifically, the decidual adjacent interchorionic space (CT-V2)-derived stem cells of the present invention have negative surface factor expression characteristics for CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4; Or CD49b, CD74 or CD200 may be characterized as having a positive surface factor expression characteristic, preferably one or more selected from the group consisting of CD24, CD104, CD227, Disialogangolioside GD2 and SSEA-4, two or more, Three or more, four or more, and all five surface factors may be all negative, and/or one or more, two or more, or all three surface factors selected from the group consisting of CD49b, CD74 and CD200 may all be positive.
이러한 표면 인자 발현 특성은 전체 태반, 전체 융모간강 및 융모막판 인접 융모간강 유래 줄기세포와는 구별되는 본 발명 탈락막 인접 융모간강(CT-V2) 유래 줄기세포만의 독특한 표면인자 발현 특성이며, 특히 전체 태반, 전체 융모간강 및 융모막판 인접 융모간강 유래 줄기세포와 달리 CD49b 및 CD200 표면인자 발현이 높고, CD24 및 CD227 에 대하여 음성의 표면마커 발현 패턴을 나타낸다는 점에서 본 발명의 줄기세포가 새롭게 분리된 줄기세포임을 확인하였다. This surface factor expression characteristic is a unique surface factor expression characteristic of the stem cells derived from the decidua adjacent interchorionic space (CT-V2) of the present invention, which is distinguished from stem cells derived from the whole placenta, the entire interchorionic cavity, and the chorionic plate adjacent interchorionic space. Unlike stem cells derived from the whole placenta, the entire interchorionic cavity and the chorionic plate adjacent interchorionic space, the stem cells of the present invention are newly isolated in that they show high expression of CD49b and CD200 surface factors and negative surface marker expression patterns for CD24 and CD227. It was confirmed that the stem cells were
또한 본 발명의 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 또는 SSEA-4에 대하여 음성의 표면인자 발현; 및 CD44, CD73, CD90, CD105, CD49b, CD74 또는 CD200 에 대하여 양성의 표면인자 발현 특성을 가질 수 있고, 상기 CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 및 SSEA-4 로 이루어진 군에서 선택된 1종 이상, 2종 이상, 3종 이상, 4종 이상, 5종 이상, 6종 이상, 7종 이상, 8종 이상, 9종 표면인자가 모두 음성이고/이거나, CD44, CD73, CD90, CD105, CD49b, CD74 및 CD200 로 이루어진 군에서 선택된 1종 이상, 2종 이상, 3종 이상, 4종 이상, 5종 이상, 6종 이상, 7종 표면인자가 모두 양성일 수 있다. In addition, the decidual adjacent interchorionic space (CT-V2)-derived stem cells of the present invention CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4 negative surface factor expression; and positive surface factor expression properties for CD44, CD73, CD90, CD105, CD49b, CD74 or CD200, the CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 and SSEA-4 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 surface factors selected from the group consisting of are all negative, and / or CD44, One or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, and 7 surface factors selected from the group consisting of CD73, CD90, CD105, CD49b, CD74 and CD200 may all be positive.
본 발명에서 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 모체 세포 기원의 줄기세포일 수 있다. 태반은 태아와 모체 유래의 특성을 나타내는 고유한 조직으로 자궁에 붙어있는 기저막, 태반의 중심조직으로 모체와 태아 혈액간에 물질 교환을 매개하는 융모조직, 태반의 안쪽으로 태반을 덮고 있으면서 태아와 양막을 싸고있는 융모막 등 개별적 특성을 나타내는 조직들로 이루어져 있다. 따라서 태반 유래 줄기세포라고 할지라도 수득 위치에 따라 세포의 기원이 상이하게 나타날 수 있으며, 영양막 유래 줄기세포는 모체 세포 기원, 융모 유래 줄기세포는 태아 세포 기원, 전체 태반 유래 줄기세포의 경우 태아 및 모체 모두에서 기원하는 줄기세포인 것으로 알려져 있다. 본 발명의 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 염색체 형 분석을 통해 확인한 결과 모체 유래의 세포 염색체 형을 갖는 것을 확인하였다In the present invention, the stem cells derived from the decidua adjacent interchorionic space (CT-V2) may be stem cells derived from parental cells. The placenta is a unique tissue that shows the characteristics of the fetus and the mother. The basement membrane attached to the uterus, the villous tissue that mediates the exchange of substances between the mother and the fetal blood as the central tissue of the placenta, and the fetus and the amniotic membrane while covering the placenta inside It consists of tissues showing individual characteristics, such as the chorion surrounding it. Therefore, even placental-derived stem cells may have different origins depending on the location where they are obtained, trophoblast-derived stem cells are of maternal origin, villous-derived stem cells are fetal cells, and whole placental-derived stem cells are fetal and maternal. It is known that stem cells originate from both. The stem cells derived from the decidua adjacent interchorionic space (CT-V2) of the present invention were confirmed to have a maternal-derived cell chromosome type as a result of chromosomal analysis.
본 발명에서 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 당 분야에 알려진 방법을 제한없이 사용하여 수득될 수 있고 바람직하게는 1) 전체 영양막층 중 탈락막 인접 부위에서부터 2/3 지점까지인 영양막층 부위를 절단하여 탈락막 인접 영양막층을 얻는 단계; 2) 상기 탈락막 인접 영양막층에서 융모를 제거하여 탈락막 인접 융모간강을 얻는 단계; 및 3) 상기 탈락막 인접 융모간강에서 줄기세포를 수득하는 단계; 를 통해 수득되는 것을 특징으로 하는; 를 통해 수득 될 수 있다. In the present invention, stem cells derived from the interchorionic space (CT-V2) adjacent to the decidua can be obtained using any method known in the art without limitation, and preferably 1) from the region adjacent to the decidua in the entire trophoblast layer to 2/3 points cutting the phosphorus trophoblast layer to obtain a trophoblast layer adjacent to the decidua; 2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; Characterized in that obtained through; can be obtained through
따라서 본 발명은 1) 전체 영양막층 중 탈락막 인접 부위에서부터 2/3 지점까지인 영양막층 부위를 절단하여 탈락막 인접 영양막층을 얻는 단계; 2) 상기 탈락막 인접 영양막층에서 융모를 제거하여 탈락막 인접 융모간강을 얻는 단계; 및 3) 상기 탈락막 인접 융모간강에서 줄기세포를 수득하는 단계; 를 포함하는 탈락막 인접 융모간강 유래 줄기세포의 분리 수득 방법을 제공한다. Therefore, the present invention comprises the steps of 1) obtaining a trophoblast adjacent to the decidua by cutting the portion of the trophoblast that is from the portion adjacent to the decidua to the 2/3 point among the entire trophoblast layer; 2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; It provides a method for separating and obtaining stem cells derived from the interchorionic space adjacent to the decidua, comprising a.
상기 1) 단계는 태반으로부터 분리된 전체 영양막층 중 탈락막 인접 영양막층을 분리하는 단계로, 전체 영양막을 3등분 하였을 때, 탈락막 인접 부위에서부터 2/3 지점까지의 연속적인 부위를 절단하여 탈락막 인접 영양막층을 얻는 것을 특징으로 한다. Step 1) is a step of separating the trophoblast adjacent to the decidua out of the entire trophoblast layer separated from the placenta. When the entire trophoblast is divided into three, a continuous portion from the adjacent portion to the decidua to 2/3 points is cut and dropped off. Characterized in obtaining a membrane adjacent trophoblast layer.
상기 2) 단계는 융모와 융모간강이 함께 존재하는 탈락막 인접 영양막층에서 융모를 제거하여 순수한 융모간강만을 분리하는 단계를 의미한다. 2) 단계 이후 탈락막 인접 융모간강을 PBS 로 세척하여 남아있는 혈액 및 혈구세포를 제거하는 단계를 추가적으로 포함할 수 있다. In step 2), the villi are removed from the trophoblast layer adjacent to the decidua in which the villi and the interchorionic cavity exist together, and thus only the pure interchorionic cavity is separated. After step 2), the method may further include washing the interchorionic cavity adjacent to the decidua with PBS to remove the remaining blood and blood cells.
이후 3) 단계는 탈락막 인접 융모간강 조직으로부터 줄기세포를 수득하는 단계로 탈락막 인접 융모간강 조직에 효소 용액을 가하여 효소반응을 수행하여 얻어진 세포를, 성장 인자를 사용하지 않고, 우태아 혈청 및 항생제가 첨가된 배지에서 배양한 다음 회수함으로써 수득될 수 있다. 상기 효소에는 트립신(Trypsin), 콜라게나아제(collagenase), 디스파아제(dispase), DNase, RNase, 프로테아제(protease), 리파아제(lipase), 히알루로니다아제(hyaluronidase) 및 엘라스타제 (elastase) 등이 포함되나, 이에 제한되지 않는다. 상기 콜라게나아제는 콜라게나아제 A, I, II, III 또는 IV 등을 포함할 수 있다. After that, step 3) is a step of obtaining stem cells from the interchorionic space tissue adjacent to the decidua. The cells obtained by adding an enzyme solution to the interchorionic space tissue adjacent to the decidua and performing an enzymatic reaction, without using growth factors, are treated with fetal bovine serum and It can be obtained by culturing in an antibiotic-added medium and then recovering. The enzymes include trypsin, collagenase, dispase, DNase, RNase, protease, lipase, hyaluronidase and elastase. and the like, but are not limited thereto. The collagenase may include collagenase A, I, II, III or IV, and the like.
상기와 같은 방법으로 분리된 탈락막 인접 융모간강 유래 줄기세포는 CD24, CD104, CD227, Disialogangolioside GD2 또는 SSEA-4 에 대하여 음성의 표면인자 발현 특성을 갖거나; 또는 CD49b, CD74 또는 CD200에 대하여 양성의 표면인자 발현 특성을 갖는 것을 특징으로 할 수 있고 바람직하게는 CD24, CD104, CD227, Disialogangolioside GD2 및 SSEA-4로 이루어진 군에서 선택된 1종 이상, 2종 이상, 3종 이상, 4종 이상, 5종 표면인자가 모두 음성이고/이거나 CD49b, CD74 및 CD200로 이루어진 군에서 선택된 1종 이상, 2종 이상, 3종 표면인자가 모두 양성일 수 있다. The decidual-proximal interchorionic space-derived stem cells isolated by the above method have negative surface factor expression characteristics for CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4; Or CD49b, CD74 or CD200 may be characterized as having a positive surface factor expression characteristic, preferably one or more selected from the group consisting of CD24, CD104, CD227, Disialogangolioside GD2 and SSEA-4, two or more, Three or more, four or more, and five surface factors may all be negative, and/or one or more, two or more, and three surface factors selected from the group consisting of CD49b, CD74 and CD200 may all be positive.
본 발명에 따른 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 서로 다른 종류의 세포로 분화할 수 있으며, 예를 들어, 지방세포, 연골세포, 골세포, 신경세포, 인대세포 또는 건세포(tenocyte) 등 다양한 종류의 세포로 분화할 수 있고, 이에 제한되지 않는다. The stem cells derived from the decidual interchorionic space (CT-V2) according to the present invention can be differentiated into different types of cells, for example, adipocytes, chondrocytes, osteocytes, nerve cells, ligament cells, or tendon cells. (tenocyte) may be differentiated into various types of cells, but is not limited thereto.
따라서 본 발명은 탈락막 인접 융모간강(CT-V2) 유래 줄기세포를 유효성분으로 포함하는 조직 재생용 세포치료제 및 조직 재생용 조성물을 제공한다. Accordingly, the present invention provides a cell therapy agent for tissue regeneration and a composition for tissue regeneration comprising stem cells derived from the decidua adjacent interchorionic space (CT-V2) as an active ingredient.
또한 본 발명은 탈락막 인접 융모간강 유래 줄기세포를 이를 필요로 하는 개체에 처리하는 단계; 를 포함하는 조직재생 방법으로, 상기 탈락막 인접 융모간강은 전체 영양막 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인, 조직 재생방법을 제공한다. In addition, the present invention comprises the steps of treating a subject in need thereof with stem cells derived from the chorionic space adjacent to the decidua; In a tissue regeneration method comprising a, the interchorionic cavity adjacent to the decidua provides a tissue regeneration method, which is a trophoblast of the entire trophoblast adjacent to the decidua to 2/3 of the total trophoblast.
본 발명에서 "분화(differentiation)"란, 일반적으로 비교적 단순 한계가 둘 이상의 질적으로 다른 부분계로 분리되는 현상을 의미하는데, 구체적으로 세포가 분열 증식하여 성장하는 동안에 서로 구조나 기능이 특수화하는 현상, 즉 생물의 세포, 조직 등이 각각에게 주어진 일을 수행하기 위하여 형태나 기능이 변해가는 현상을 의미한다. 상대적으로, "미분화"란 상술한 분화가 일어나지 않은, 아직 줄기세포로써의 특징을 함유하고 있는 상태를 의미한다.In the present invention, "differentiation" generally refers to a phenomenon in which a relatively simple limit is separated into two or more qualitatively different subsystems. In other words, it means a phenomenon in which cells, tissues, etc. of living things change form or function in order to perform a given task. Relatively, "undifferentiated" refers to a state in which the above-described differentiation has not occurred and yet contains the characteristics of stem cells.
줄기세포를 분화시키는 방법은 종래 공지된 방법에 따라 수행될 수 있으며, 특별히 제한되지 않는다. 예를 들어, 상기 줄기세포를 덱사메타손(dexamethasone), 인도메타신(indomethacin), 인슐린 및 IBMX(3-isobutyl-1-methylxanthine)를 포함하는 배지에서 배양하여 지방세포로 분화시키는 방법; 상기 줄기세포를 덱사메타손, BMP-6(bone morphogenetic protein 6), TGF-βfactor beta), 아스코르브산(ascorbic acid) 및 L-프롤린(L-proline)을 포함하는 배지에서 배양하여 연골세포로 분화시키는 방법; 상기 줄기세포를 덱사메타손, 아스코르브산, β글리크로포스페이트(β및 아스코르브산-2-포스페이트(ascorbic acid-2-phosphate)를 포함하는 배지에 배양하여 골세포로 분화시키는 방법 등을 사용함이 바람직하다.The method for differentiating stem cells may be performed according to a conventionally known method, and is not particularly limited. For example, a method of culturing the stem cells in a medium containing dexamethasone, indomethacin, insulin and IBMX (3-isobutyl-1-methylxanthine) to differentiate them into adipocytes; A method of culturing the stem cells in a medium containing dexamethasone, bone morphogenetic protein 6 (BMP-6), TGF-βfactor beta, ascorbic acid and L-proline to differentiate them into chondrocytes ; It is preferable to use a method for differentiating the stem cells into osteocytes by culturing the stem cells in a medium containing dexamethasone, ascorbic acid, and β-glycrophosphate (β and ascorbic acid-2-phosphate).
상기 방법으로 분화된 탈락막 인접 융모간강 조직에서 유래된 줄기세포의 분화 정도를 측정하는 방법은 특별히 이에 제한되지 않으나, 당해 분야에 공지된 기법인 유세포 분석방법, 면역세포화학적 방법, PCR 또는 유전자-발현 프로파일을 사용하여 세포 표면 표지 또는 형태의 변화를 측정하는 방법, 광학 현미경 또는 공초점 현미경을 사용하여 세포의 형태변화를 조사하는 방법, 유전자 발현 프로파일의 변화를 측정하는 방법 등을 사용할 수 있고, 바람직하게는 RT-PCR, Oil-red O 염색법, Safranin O 염색법, Type II collagen 면역조직화학 염색법, ALP(alkaline phosphate) 염색법 또는 Alizarin red S 염색법 등을 이용할 수 있다.The method for measuring the degree of differentiation of stem cells derived from the decidual adjacent interchorionic cavity tissue differentiated by the above method is not particularly limited thereto, but is a technique known in the art such as flow cytometry, immunocytochemical method, PCR or gene- A method of measuring a change in cell surface label or morphology using an expression profile, a method of examining a morphological change of a cell using an optical microscope or a confocal microscope, a method of measuring a change in a gene expression profile, etc. can be used, Preferably, RT-PCR, Oil-red O staining, Safranin O staining, Type II collagen immunohistochemical staining, ALP (alkaline phosphate) staining, or Alizarin red S staining can be used.
본 발명에서 "세포치료제(cellular therapeutic agent)"란, 인간으로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA 규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 이러한 세포가 질병의 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다. In the present invention, the term "cellular therapeutic agent" refers to cells and tissues isolated from humans, cultured, and manufactured through special manipulation, and as pharmaceuticals (U.S. FDA regulations) used for the purpose of treatment, diagnosis, and prevention. Through a series of actions, such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways to restore the function of tissues, these cells are used for the purpose of treatment, diagnosis and prevention of diseases medicines used as
본 발명의 탈락막 인접 융모간강 조직 유래 줄기세포는 신체의 조직 또는 기관이 목적하는 세포 군집, 예를 들어 줄기세포 또는 분화세포 군집의 생착, 이식 또는 주입에 의해 조정, 강화, 치료 또는 대체되는 다양한 종류의 치료 프로토콜에 사용될 수 있다. 본 발명의 융모간강 조직 유래 줄기세포는 존재하는 조직을 대체 또는 강화시켜, 새롭거나 변화된 조직이 되게 하거나 생물학적 조직 또는 구조와 결합시킬 수 있다. The stem cells derived from the decidua proximal interchorionic cavity tissue of the present invention are various types of tissue or organs in the body that are modulated, enhanced, treated or replaced by engraftment, transplantation, or injection of a desired cell population, for example, a stem cell or differentiated cell population. It can be used for any kind of treatment protocol. Interchorionic cavity tissue-derived stem cells of the present invention can replace or strengthen existing tissues, resulting in new or altered tissues, or can be combined with biological tissues or structures.
바람직하게는 본 발명의 세포치료제는 지방, 연골, 골, 신경, 인대 및 건으로 이루어진 군에서 선택된 1종 이상의 조직을 재생하기 위한 용도로 사용될 수 있다. 또한 본 발명에 있어 연골은 유리연골, 섬유 연골 또는 탄성연골을 제한없이 포함할 수 있으며, 상기 연골은 관절연골(articular Cartilage), 귀 연골, 비연골, 팔꿈치 연골, 반월상연골(meniscus), 무릎연골, 늑연골, 발목연골, 관연골, 후두연골 및 척추 연골로 이루어진 군으로부터 선택되는 것을 특징으로 할 수 있다. Preferably, the cell therapy agent of the present invention can be used for regenerating one or more tissues selected from the group consisting of fat, cartilage, bone, nerve, ligament and tendon. In addition, in the present invention, the cartilage may include, without limitation, free cartilage, fibrocartilage or elastic cartilage, and the cartilage is articular cartilage, ear cartilage, noncartilage, elbow cartilage, meniscus, knee. It may be characterized in that it is selected from the group consisting of cartilage, cost cartilage, ankle cartilage, cartilage cartilage, occipital cartilage and vertebral cartilage.
따라서 본 발명의 세포치료제는 골 결손, 건-인대 결손, 지방조직 결손, 연골 괴사, 골연골염, 연골파열, 연골외상, 관절염, 연골결핍 또는 선천성 기관연화 치료용일 수 있다. Therefore, the cell therapeutic agent of the present invention may be used for treating bone defect, tendon-ligament defect, adipose tissue defect, cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma, arthritis, cartilage deficiency or congenital organ softening.
또한, 본 발명의 세포치료제 또는 조직 재생용 조성물은 관절 내 또는 관절 강에 직접 투여될 수 있으며, 이 경우 주사제 형태로 투여될 수 있다. 본 발명의 탈락막 인접 융모간강 유래 줄기세포는 연골, 골, 지방 등으로 분화될 수 있는 분화능이 뛰어난 줄기세포이므로, 관절, 연골, 건 또는 인대 부위에 투여함으로써, 다양한 병변, 예컨대 관절 연골의 병변을 치료하거나 예방할 수 있다. 특히 본 발명의 탈락막 인접 융모간강 유래 줄기세포를 관절 내 투여하면 연골 표면을 부드럽고 손상이 거의 없는 상태로 재생시킬 수 있으며, 관절염이 진행되지 않도록 연골을 보호 및 재생할 수 있다. In addition, the cell therapy agent or the composition for tissue regeneration of the present invention may be administered directly into a joint or joint cavity, and in this case, it may be administered in the form of an injection. Since the stem cells derived from the decidua adjacent chorionic space of the present invention are stem cells with excellent differentiation ability that can be differentiated into cartilage, bone, fat, etc., they can be administered to joints, cartilage, tendons, or ligaments by administering to various lesions, such as lesions of articular cartilage. can be treated or prevented. In particular, intra-articular administration of the stem cells derived from the decidua adjacent chorionic space of the present invention can regenerate the cartilage surface in a soft and almost free state, and protect and regenerate cartilage so that arthritis does not progress.
특히 본 발명의 세포 치료제 또는 조직 재생용 조성물은 탈락막 인접 융모간강 유래 줄기세포를 직접 투여하기 위하여 주사제 형태로 제형화 될 수 있으며, 이 경우, 주사제에 적합하도록 하이드로겔을 더 포함할 수 있다. In particular, the composition for cell therapy or tissue regeneration of the present invention may be formulated in the form of an injection for direct administration of stem cells derived from the decidua adjacent chorionic space, and in this case, may further include a hydrogel suitable for injection.
본 발명의 세포 치료제에 사용할 수 있는 하이드로겔은 주사제에 적합한 당 분야에 공지된 하이드로겔을 제한없이 포함할 수 있으며, 소장점막하 조직, 히알루론산(hyalurinic acid), 카복시메틸셀룰로오스(CMC, carboxymethylcellulose), 알지네이트(alginate), 키토산(chitosan), 폴리아크릴아미드(polyacrylamide), 폴리(N-이소프로필아크릴아미드)(poly(N-isopropylacrylamide)), β글리세로포스페이트(β플루로닉(Poly(ethylene oxide)poly(propylene oxide)poly(ethyleneoxide), Pluronic), 피브린, 폴리에틸렌옥사이드 (PEO) 및 카복시메틸셀룰로오스(CMC)와 폴리에틸렌이민(PEI)의 혼합물로 이루어진 군에서 선택된 1종 이상일 수 있고, 본 발명의 바람직한 실시예에서는 히알루론산, 보다 구체적으로 히알루론산 나트륨을 탈락막 인접 융모간강 유래 줄기세포와 혼합하여 사용하였다. The hydrogel that can be used in the cell therapy of the present invention may include, without limitation, a hydrogel suitable for injection, known in the art, small intestine submucosal tissue, hyaluronic acid, carboxymethylcellulose (CMC, carboxymethylcellulose), Alginate, chitosan, polyacrylamide, poly (N-isopropylacrylamide) (poly (N-isopropylacrylamide)), β glycerophosphate (β pluronic (Poly (ethylene oxide)) poly(propylene oxide) poly(ethyleneoxide), Pluronic), fibrin, polyethylene oxide (PEO) and at least one selected from the group consisting of a mixture of carboxymethyl cellulose (CMC) and polyethyleneimine (PEI), preferred of the present invention In the Examples, hyaluronic acid, more specifically sodium hyaluronate, was used by mixing with stem cells derived from the decidua adjacent interchorionic space.
본 발명의 세포치료제의 바람직한 투여량은 개체의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있으며, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the cell therapy agent of the present invention varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. Administration may be administered once a day or may be administered in several divided doses, and the dosage is not intended to limit the scope of the present invention in any way.
본 발명의 탈락막 인접 융모간강 유래 줄기세포 또는 이를 포함하는 세포 치료제의 처리, 투여 대상이 되는 개체는 지방, 연골, 골, 신경, 인대 및 건으로 이루어진 군에서 선택된 1종 이상의 조직이 손상, 결실되어 조직 재생이 필요하거나, 필요성이 예상되는 개체일 수 있다. 본 발명의 세포치료제를 개체에 투여하는 경우 줄기세포 치료제의 효과를 달성하고, 개체 내에서 조직 재생을 효과적으로 유도할 수 있는 한 당 분야에 공지된 제형으로 제한없이 사용할 수 있으며, 바람직하게는 주사제 형태로 제형화하여 투여될 수 있다. The subject subject to treatment and administration of the decidual-proximal interchorionic space-derived stem cell or cell therapeutic agent containing the same of the present invention is damaged or deleted at least one tissue selected from the group consisting of fat, cartilage, bone, nerve, ligament, and tendon. It may be an individual in need or foreseeable need for tissue regeneration. When the cell therapy agent of the present invention is administered to an individual, it can be used without limitation as a formulation known in the art as long as it can achieve the effect of the stem cell therapy and effectively induce tissue regeneration in the individual, preferably in the form of an injection. It can be formulated and administered.
본 발명에 따른 탈락막 인접 융모간강 유래 줄기세포는 증식능과 분화능이 우수하여 조직 재생 효과가 우수하며, 조직 재생용 세포 치료제에서 기술된 내용과 중복되는 내용은 명세서 기재의 복잡성을 피하기 위해 생략한다. The stem cell derived from the decidua adjacent chorionic space according to the present invention has excellent proliferation and differentiation ability and thus has excellent tissue regeneration effect, and the content overlapping with the content described in the cell therapy for tissue regeneration is omitted to avoid the complexity of the description of the specification.
이하, 하기 실시예를 통하여 본 발명을 보다 상세히 설명한다. 이들 실시예는 본 발명을 상세히 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. These examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited by these examples.
실시예 1: 태반 탈락막 인접 융모간강 분리 및 줄기세포 수득Example 1: Separation of the interchorionic cavity adjacent to the placental decidua and obtaining stem cells
태반 조직으로부터 양막을 벗겨 내고, 탈락막을 멸균된 가위를 이용하여 제거한 후, 약 5*5*1.5cm 크기의 탈락막 인접 영양막층(chorion)을 얻어 150㎜ 디쉬에 옮긴 후, PBS를 이용하여 5번이상 세척하여 혈액 및 혈구세포를 제거하였다. 탈락막 인접 영양막층은 전체 영양막 중 탈락막 쪽에 인접한 영양막층으로, 전체 영양막을 3등분 하였을 때, 탈락막 인접 부위에서부터 2/3 지점까지의 영양막층으로 정의하였다. 탈락막 인접 영양막 충에서 융모간강을 분리해 내기 위해, 탈락막 인접 영양막층을 융모와 intervillous로 다시 분리한 후, 융모를 제거하여 분리된 융모간강을 PBS를 이용하여 남아있는 혈액 및 혈구세포를 제거하기 위해 5번 이상 세척하였다. 이렇게 얻은, 탈락막 인접 영양막층에서 분리된 융모간강 조직을 전체 융모간강(intervillous space, IVS) 조직과 구별하기 위하여 '탈락막 인접 융모간강(CT-V2)' 으로 정의하였다. 탈락막 인접 융모간강을 도식화하여 도 1에 나타내었다. After peeling off the amniotic membrane from the placental tissue, removing the decidua using sterile scissors, obtaining a chorion adjacent to the decidua with a size of about 5*5*1.5cm and transferring it to a 150mm dish, using PBS for 5 After washing more than once, blood and blood cells were removed. The trophoblast layer adjacent to the decidua was defined as the trophoblast layer adjacent to the decidua among the total trophoblasts, and when the entire trophoblast was divided into thirds, it was defined as the trophoblast layer from the area adjacent to the decidua to the 2/3 point. To isolate the interchorionic cavity from the trophoblast adjacent to the decidua, the trophoblast layer adjacent to the decidua is again separated into villi and intervillous, and then the villi are removed. It was washed more than 5 times to In order to distinguish the obtained interchorionic space tissue separated from the trophoblast layer adjacent to the decidua from the entire intervillous space (IVS) tissue, it was defined as 'the decidua adjacent interchorionic space (CT-V2)'. A schematic diagram of the interchorionic cavity adjacent to the decidua is shown in FIG. 1 .
탈락막 인접 융모간강 (이하, CT-V2) 조직을 50ml 튜브에 옮긴 후, 0.2% 콜라게나아제를 첨가한 a-MEM 배지를 가하고 37℃ 조건에서 교반기를 이용하여 2시간 정도 반응시켜 CT-V2 로부터 유래된 세포를 수득하였다. 수득한 CT-V2로부터 유래된 세포를 100 ㎛ 메쉬에 여과하여 분해되지 않은 조직을 제거하고, 우태아 혈청 및 항생제가 첨가된 a-MEM 배지를 가한 후 25℃, 1500rpm에서 4분간 원심 분리하였다. 상층액을 제거하고 남은 침전된 세포에 성장인자를 포함하지 않고, 우태아 혈청 및 항생제가 첨가된 a-MEM 배지를 가하고, 37℃, 5% CO2 조건하에서 배양하였다. 상기 배양물에서 배양 용기의 바닥에 부착된 세포를 선별하여 CT-V2 로부터 유래된 줄기세포를 수득하였다. After transferring the tissue of the interchorionic space (hereinafter, CT-V2) adjacent to the decidua to a 50 ml tube, a-MEM medium supplemented with 0.2% collagenase was added, and the mixture was reacted for 2 hours using a stirrer at 37 ° C. Cells derived from The obtained CT-V2 derived cells were filtered through a 100 μm mesh to remove undecomposed tissue, and a-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25° C. and 1500 rpm for 4 minutes. After removing the supernatant, a-MEM medium containing no growth factors and containing fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at 37° C. under 5% CO 2 conditions. In the above culture, cells attached to the bottom of the culture vessel were selected to obtain stem cells derived from CT-V2.
비교예 1: 타 조직 유래 줄기세포의 수득Comparative Example 1: Obtaining stem cells derived from other tissues
1-1. 태반 전체로부터 유래된 줄기세포의 수득1-1. Obtaining stem cells derived from whole placenta
전체 태반 조직을 약 5*5*3cm 정도를 세절하고, PBS로 세척하여 태반 조직으로부터 혈액 및 혈구세포를 제거하였다. 상기 세척된 태반 조직에 0.2% 콜라게나아제를 첨가한 a-MEM 배지를 가하고 37℃ 조건에서 교반기를 이용하여 반응시켜 태반 세포를 수득하였다. 상기 수득한 태반 세포를 100 ㎛ 메쉬에 여과하여 분해되지 않은 조직을 제거하고, 우태아 혈청 및 항생제가 첨가된 a-MEM 배지를 가한 후 25℃, 1000rpm에서 4분간 원심 분리하였다. 상층액을 제거하고 남은 침전된 세포에 성장인자를 포함하지 않고, 우태아 혈청 및 항생제가 첨가된 a-MEM 배지를 가하고, 37℃, 5% CO2 조건하에서 배양하였다. 상기 배양물에서 배양 용기의 바닥에 부착된 세포를 선별하여 태반 전체(Whole placenta, Pla) 유래 줄기세포를 수득하였다.The whole placental tissue was cut into about 5*5*3cm and washed with PBS to remove blood and blood cells from the placental tissue. A-MEM medium supplemented with 0.2% collagenase was added to the washed placental tissue and reacted using a stirrer at 37° C. to obtain placental cells. The obtained placental cells were filtered through a 100 μm mesh to remove undecomposed tissue, and a-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25° C. and 1000 rpm for 4 minutes. After removing the supernatant, a-MEM medium containing no growth factors and containing fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at 37° C. under 5% CO 2 conditions. Cells attached to the bottom of the culture vessel were selected from the culture to obtain stem cells derived from whole placenta (Pla).
1-2. 태반 세부조직으로부터 유래된 줄기세포의 수득1-2. Obtaining Stem Cells Derived from Placental Details
태반 조직으로부터 양막을 벗겨내고 탈락막을 제거한 뒤, 약 5*5*3cm 크기의 영양막층 전체를 얻어 150㎜ 디쉬에 옮긴 후, PBS를 이용하여 5번이상 세척하여 혈액 및 혈구세포를 제거하였다. 융모를 제외한 전체 융모간강(intervillous space, IVS) 만을 분리해 내기 위해, 영양막 전체를 융모와 intervillous 로 다시 분리한 후, 분리된 intervillous 를 PBS를 이용하여 남아있는 혈액 및 혈구세포를 제거하기 위해 5번 이상 세척하였다. After peeling off the amniotic membrane from the placental tissue and removing the decidua, the entire trophoblast layer with a size of about 5*5*3cm was obtained and transferred to a 150mm dish, and washed more than 5 times with PBS to remove blood and blood cells. In order to separate only the entire intervillous space (IVS) except for the villi, the entire trophoblast was separated again into villi and intervillous, and then the separated intervillous was used 5 times to remove the remaining blood and blood cells using PBS. It was washed over.
전체 융모간강(IVS) 에서 실시예 1에 기재된 탈락막 인접 융모간강 (CT-V2) 을 제외한 나머지 조직 부분을 CT-V1 으로 정의하였다. 전체 융모간강 또는 CT-V1 조직을 50ml 튜브에 옮긴 후, 0.2% 콜라게나아제를 첨가한 a-MEM 배지를 가하고 37℃ 조건에서 교반기를 이용하여 2시간 정도 반응시켜 각각의 조직으로부터 유래된 세포를 수득하였다. 수득한 세포는 100 ㎛ 메쉬에 여과하여 분해되지 않은 조직을 제거하고, 우태아 혈청 및 항생제가 첨가된 a-MEM 배지를 가한 후 25℃, 1500rpm에서 4분간 원심 분리하였다. 상층액을 제거하고 남은 침전된 세포에 성장인자를 포함하지 않고, 우태아 혈청 및 항생제가 첨가된 a-MEM 배지를 가하고, 37℃, 5% CO2 조건하에서 배양하였다. In the total interchorionic space (IVS), the remaining tissue part except for the decidual adjacent interchorionic space (CT-V2) described in Example 1 was defined as CT-V1. After transferring the entire interchorionic cavity or CT-V1 tissue to a 50ml tube, a-MEM medium supplemented with 0.2% collagenase was added, and the cells derived from each tissue were reacted for 2 hours at 37°C using a stirrer. obtained. The obtained cells were filtered through a 100 μm mesh to remove undecomposed tissue, and a-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25° C. and 1500 rpm for 4 minutes. After removing the supernatant, a-MEM medium containing no growth factors and containing fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at 37° C. under 5% CO 2 conditions.
실시예 2: 탈락막 인접 융모간강(CT-V2) 유래 줄기세포 배양 및 세포 형태 확인Example 2: Decidual adjacent interchorionic space (CT-V2) derived stem cell culture and cell morphology confirmation
상기 실시예 1 에서 수득한 CT-V2 유래 줄기세포를 PBS로 세척한 후, 성장인자를 포함하지 않고, 우태아 혈청 및 항생제가 첨가된 a-MEM 배지를 2~3일마다 교체하면서 배양하였다. 상기 줄기세포가 80% 이상 성장한 시점에서 트리플(TryPLE)을 처리하여 줄기세포를 배양 용기에서 분리하고, 분리된 줄기세포를 1/5의 비율로 희석한 다음, 다른 배양 용기에서 배양하는 방법으로 계대 배양을 수행하였다. 배양한 이후의 세포 형태를 현미경으로 관찰하였다.After washing the CT-V2-derived stem cells obtained in Example 1 with PBS, the a-MEM medium containing no growth factors, fetal bovine serum and antibiotics was added and cultured while replacing every 2-3 days. When the stem cells have grown more than 80%, the stem cells are separated from the culture vessel by treating the triple (TryPLE), the separated stem cells are diluted at a ratio of 1/5, and then passaged by culturing in another culture vessel. Incubation was performed. Cell morphology after culture was observed under a microscope.
또한, 상기 비교예 1에서 수득한 태반 전체(Whole placenta, Pla) 및 다른 세부 조직 유래 줄기세포를 이용하여, 동일한 방법으로 계대 배양을 수행한 후, 배양한 이후의 세포 형태를 현미경으로 관찰하였다. 그 결과를 각 도 2 에 나타내었다. In addition, using the whole placenta (Pla) and other detailed tissue-derived stem cells obtained in Comparative Example 1, subculture was performed in the same manner, and the cell morphology after culturing was observed under a microscope. The results are shown in each FIG. 2 .
도 2에 나타낸 바와 같이, 본 발명에 따른 CT-V2 유래 줄기세포는 섬유아세포 모양의 형태적 특성을 나타내고 단일의 세포만을 특이적으로 유지하는 것을 확인할 수 있었다. 반면 태반 전체(Whole placenta, Pla) 및 다른 세부 조직유래 줄기세포는 섬유아세포 모양의 형태적 특성을 나타냈으나, 다수의 서로 다른 형태의 세포가 혼합되어 있는 것을 확인할 수 있었다. As shown in FIG. 2 , it was confirmed that the CT-V2-derived stem cells according to the present invention exhibited morphological characteristics of fibroblasts and specifically maintained only single cells. On the other hand, whole placenta (Pla) and other detailed tissue-derived stem cells showed fibroblast-like morphological characteristics, but it was confirmed that a number of different types of cells were mixed.
즉, 본 발명에 따른 CT-V2 유래 줄기세포는 계대 배양 전후로 단일 세포만을 특이적으로 유지하였으나, 태반 전체 및 다른 세부 조직 유래 줄기세포로부터 유래된 줄기세포는 서로 다른 형태의 세포가 혼합되어 있음을 확인하였다. That is, the CT-V2-derived stem cells according to the present invention specifically maintained only single cells before and after subculture, but stem cells derived from stem cells derived from the whole placenta and other detailed tissues were mixed with cells of different types. Confirmed.
실시예 3: 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 집락형성능 분석Example 3: Analysis of colony formation performance of stem cells derived from the decidua adjacent interchorionic space (CT-V2)
상기 실시예 1에서 수득한 CT-V2 유래 줄기세포의 집락형성능을 확인하였다. 보다 구체적으로, 상기 실시예 1에서 수득한 CT-V2 유래 줄기세포를 상기 실시예 2의 방법으로 첫 번째 계대 배양을 수행하고, 상기 계대 배양이 종료되는 시점에서 100㎜ 디쉬에 5 X 103개씩 접종(seeding)한 후, 14일 동안 성장인자를 포함하지 않고 우태아 혈청 및 항생제가 첨가된 a-MEM 배지에서 배양하였다. 그 후 각 줄기세포에서 몇 개의 집락이 형성되는지를 계수하였다. 또한, 상기 비교예 1에서 수득한 태반 전체 및 다른 태반 세부조직 유래 줄기세포를 이용하여, 동일한 방법으로 집단 배가 시간 및 집락형성능을 측정하였다. 집락형성능의 경우 태반 전체 유래 줄기세포의 결과값을 100%로 하여 환산하였다. 그 결과를 도 3 및 도 4 에 나타내었다. The colony-forming ability of the CT-V2-derived stem cells obtained in Example 1 was confirmed. More specifically, the first subculture of the CT-V2-derived stem cells obtained in Example 1 was performed by the method of Example 2, and 5 X 10 3 each in a 100 mm dish at the end of the subculture. After inoculation (seeding), it was cultured in a-MEM medium supplemented with fetal bovine serum and antibiotics without growth factors for 14 days. Thereafter, the number of colonies formed in each stem cell was counted. In addition, using the whole placenta obtained in Comparative Example 1 and stem cells derived from other placental detailed tissues, population doubling time and colony forming ability were measured in the same manner. In the case of colony-forming ability, the result value of whole placental-derived stem cells was converted into 100%. The results are shown in FIGS. 3 and 4 .
도 3에 나타낸 바와 같이, CT-V2 유래 줄기세포는 태반 전체 (Pla), 전체 융모간강 유래 (IVS), 전체 융모간강에서 탈락막 인접 융모간강을 제외한 융모막판 인접 융모간강(CT-V1) 유래 줄기세포와 비교하여 집락 형성능이 현저하게 우수한 것을 확인하였다. As shown in FIG. 3, CT-V2-derived stem cells are derived from the entire placenta (Pla), the entire interchorionic cavity (IVS), and the chorionic plate adjacent interchorionic cavity (CT-V1) excluding the decidual adjacent interchorionic space from the entire interchorionic cavity. It was confirmed that the colony forming ability was significantly superior to that of stem cells.
이를 통해 CT-V2 유래 줄기세포는 CT-V1 유래 줄기세포와 비교하여 2배 이상의 집락형성능을 나타내고, 전체 융모 및 다른 세부조직 유래 줄기세포와 비교하여 집락 형성능이 매우 뛰어난 줄기세포임을 확인하였다. Through this, it was confirmed that CT-V2-derived stem cells exhibited more than double the colony-forming ability compared to CT-V1-derived stem cells, and that they were very excellent in colony-forming ability compared to whole villi and other detailed tissue-derived stem cells.
또한 도 4에 나타낸 바와 같이, 집단 배가시간은 P2 까지는 비슷하게 유지되었으나, P2 이후에는 본 발명의 CT-V2 유래 줄기세포가 가장 높은 cumulative population doubling level을 나타내어, 가장 낮은 집단 배가 시간을 나타냄을 확인하였다. 이러한 결과를 통해 융모막판 인접 융모(CT-V2)로부터 유래된 줄기세포가 우수한 증식능을 가진 줄기세포임을 확인하였다 Also, as shown in FIG. 4, the population doubling time was maintained similarly until P2, but after P2, the CT-V2-derived stem cells of the present invention exhibited the highest cumulative population doubling level, indicating the lowest population doubling time. . Through these results, it was confirmed that the stem cells derived from the villi (CT-V2) adjacent to the chorionic plate were stem cells with excellent proliferative capacity.
실시예 4: 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 표면 마커 분석Example 4: Analysis of surface markers of stem cells derived from decidual adjacent interchorionic space (CT-V2)
상기 실시예 1에서 수득한 CT-V2 유래 줄기세포의 면역학적 특성을 확인하기 위해 242개 Human Cell Surface Marker Screening Panel을 이용하여 표면 마커를 분석하였다. In order to confirm the immunological characteristics of the CT-V2-derived stem cells obtained in Example 1, surface markers were analyzed using 242 Human Cell Surface Marker Screening Panels.
먼저, 실시예 1에 따라 탈락막 인접 융모간강을 수득하고 이를 PBS로 세척하고, 트리플 처리한 뒤 줄기세포를 수거하여 1200rpm에서 5분 동안 원심 분리하였다. 상층액을 제거한 후 PBS로 줄기세포를 세척한 후, 1200rpm에서 5분 동안 원심 분리하였다. 상층액을 제거한 후 줄기세포를 PBS에 부유시켜 40μm cell strainer 를 통과시켜 세포 및 BD Pharmingen Stain Buffer + EDTA 를 준비하여 혼합한다. Falcon®둥근 바닥 96 웰 플레이트 (Cat. 353910) 각 well 에 100㎕씩 분주하였다 (100,000개/well). 그 후, 각 well 에 해당되는 antibody를 20㎕씩 분주하였다. 4℃에서 30분 동안 인큐베이션 한 후, 세척을 위해 BD Pharmingen Stain Buffer + EDTA 를 각 well 에 첨가한 뒤, 1200rpm에서 5분 동안 원심 분리하였다. 다시 조심스럽게 상층액을 제거한 뒤 BD Pharmingen Stain Buffer + EDTA 로 세척하고 1200rpm에서 5분 동안 원심 분리하였다. 그 후 2차 항체를 각 well 에 100㎕씩 분주하였다. 세척을 위해 BD Pharmingen Stain Buffer + EDTA 를 각 well 에 첨가한 뒤, 1200rpm에서 5분 동안 원심 분리하였고, 상등액을 제거하고, PBS를 이용하여 조심스럽게 세척 과정을 2번 반복하였다. 상등액을 제거하고, BD Pharmingen Stain Buffer + EDTA 각 well 에 150㎕씩 분주하였다. Well 당 최소 10,000 개 이상의 세포를 플로우사이토미터(FACS)를 이용해 표면 마커를 분석하였다. 또한, 동일한 방법으로 상기 비교예 1에서 수득한 태반 전체 및 다른 태반 세부조직 유래 줄기세포의 표면 마커를 분석하였다. 그 결과를 표 1, 2에 나타내었다. First, according to Example 1, the interchorionic cavity adjacent to the decidua was obtained, washed with PBS, triple-treated, and then stem cells were collected and centrifuged at 1200 rpm for 5 minutes. After removing the supernatant, the stem cells were washed with PBS, and centrifuged at 1200 rpm for 5 minutes. After removing the supernatant, the stem cells are suspended in PBS, passed through a 40 μm cell strainer, and the cells and BD Pharmingen Stain Buffer + EDTA are prepared and mixed. Falcon® round-bottom 96-well plate (Cat. 353910) 100 μl was dispensed into each well (100,000 pieces/well). After that, 20 μl of the antibody corresponding to each well was dispensed. After incubation at 4° C. for 30 minutes, BD Pharmingen Stain Buffer + EDTA was added to each well for washing, followed by centrifugation at 1200 rpm for 5 minutes. Again, the supernatant was carefully removed, washed with BD Pharmingen Stain Buffer + EDTA, and centrifuged at 1200 rpm for 5 minutes. After that, 100 μl of secondary antibody was dispensed into each well. For washing, BD Pharmingen Stain Buffer + EDTA was added to each well, centrifuged at 1200 rpm for 5 minutes, the supernatant was removed, and the washing process was carefully repeated twice using PBS. The supernatant was removed, and 150 μl of BD Pharmingen Stain Buffer + EDTA was dispensed into each well. At least 10,000 cells per well were analyzed for surface markers using a flow cytometer (FACS). In addition, the surface markers of the whole placenta obtained in Comparative Example 1 and other placental detail-derived stem cells were analyzed in the same manner. The results are shown in Tables 1 and 2.
[표 1][Table 1]
[표 2][Table 2]
표면 마커 발현량이 5% 미만인 경우 음성 마커로 분리하며, 5% 이상인 경우 그 발현량에 따라 양성 마커 중에서도 5% 이상 30% 미만인 경우 low, 30 내지 70% 미만인 경우 middle, 70% 이상인 경우 High 로 분류하였고 이러한 기준에 따라 표 1 및 2의 마커 발현값을 기준으로 정리하여 표 3 및 4 에 마커 발현을 표시하였다. If the expression level of the surface marker is less than 5%, it is classified as a negative marker, and if it is 5% or more, it is classified as low if it is 5% or more and less than 30% among positive markers, middle if it is 30 to less than 70%, and high if it is 70% or more according to the expression level. According to these criteria, the expression values of the markers in Tables 1 and 2 were summarized, and the expression of the markers was shown in Tables 3 and 4.
[표 3][Table 3]
[표 4][Table 4]
상기 표 3 및 표 4에 나타낸 바와 같이, 본 발명의 CT-V2 은 전체 태반, IVS 및 CT-V2 유래 줄기세포와 달리 CD49b, CD200 마커 발현이 높은 것으로 확인됐으며, 다른 줄기세포와 달리 CD24 및 CD227이 음성의 표면마커 발현 패턴을 나타내는 것을 확인하였다. 이러한 발현 차이를 통해 CT-V2 유래 줄기세포가 다른 태반 세부 조직 유래 줄기세포와는 완전히 다른 CD 마커 발현 특성을 나타내는 새로운 줄기세포인 것을 확인하였다. As shown in Tables 3 and 4, CT-V2 of the present invention showed high expression of CD49b and CD200 markers, unlike whole placental, IVS, and CT-V2-derived stem cells, and unlike other stem cells, CD24 and CD227 It was confirmed that this negative surface marker expression pattern was shown. Through this difference in expression, it was confirmed that the CT-V2-derived stem cells are new stem cells that exhibit completely different CD marker expression characteristics from other placental detailed tissue-derived stem cells.
또한 상기와 같은 결과는 융모 전체로부터 유래된 줄기세포들은 다양한 CD 마커 발현 패턴을 나타내는 줄기세포들이 혼재되어 있는 상태인 것을 보여주는 결과로 본원 발명과 같이 융모간강으로부터 줄기세포를 세분화하여 분리하는 경우 보다 균일한 줄기세포를 수득할 수 있다는 사실을 확인할 수 있다. In addition, the above results show that stem cells derived from the whole villi are in a state in which stem cells showing various CD marker expression patterns are mixed. It can be confirmed that one stem cell can be obtained.
실시예 5: 탈락막 인접 융모간강으로부터 유래된 줄기세포 CT-V2의 지방세포로의 분화능 확인Example 5: Confirmation of differentiation ability into adipocytes of stem cells CT-V2 derived from the interchorionic space adjacent to the decidua
실시예 1에서 수득한 융모막판 인접 융모로부터 유래된 줄기세포의 지방세포로의 분화능을 확인하기 위하여, 줄기세포를 공지된 지방세포 분화 유도 배지 1(10% FBS, 1% Anti-biotics, 1μM 덱사메타손, 20μM 인도메타신, 10 μM 인슐린, 50μM 3-isobutyl-1-methylxanthine (IBMX)가 포함된 DMEM 배지) 및 지방세포 분화 유도 배지 2(10% FBS, 1% Anti-biotics, 10μM 인슐린이 포함된 DMEM 배지)를 3~4일씩 교대로 가하여 3주 동안 배양하여 지방세포로의 분화를 유도하였다. 상기 줄기세포의 지방세포로의 분화 정도를 측정하기 위하여, 종래 공지된 방법에 따라 Oil red O 염색을 수행하였다. 또한, 동일한 방법으로 상기 비교예 1에서 수득한 태반 전체, 다른 태반 세부조직 유래 및 타 조직 유래 줄기세포의 지방세포로의 분화능을 100% 이소프로판올 용액을 처리하여 지질을 용해시켜 500 nm에서의 흡광도를 측정하였고 그 결과를 도 5 에 나타내었다. In order to confirm the differentiation ability of stem cells derived from the villi adjacent to the chorionic plate obtained in Example 1 into adipocytes, the stem cells were subjected to a known adipocyte differentiation induction medium 1 (10% FBS, 1% Anti-biotics, 1 μM dexamethasone, DMEM medium with 20 μM indomethacin, 10 μM insulin, 50 μM 3-isobutyl-1-methylxanthine (IBMX)) and adipocyte differentiation induction medium 2 (DMEM with 10% FBS, 1% Anti-biotics, 10 μM insulin) medium) was alternately added for 3 to 4 days and cultured for 3 weeks to induce differentiation into adipocytes. In order to measure the degree of differentiation of the stem cells into adipocytes, Oil red O staining was performed according to a conventionally known method. In addition, in the same manner, the ability of the whole placenta obtained in Comparative Example 1 to differentiate into adipocytes, other placental detailed tissues, and other tissues-derived stem cells was treated with 100% isopropanol solution to dissolve the lipids and measure the absorbance at 500 nm. and the results are shown in FIG. 5 .
도 5에 나타낸 바와 같이, 본 발명에 따른 CT-V2 유래 줄기세포는 IVS 와 CT-V1 유래 줄기세포에 비해 우수한 우수한 지방세포 분화능을 가지고 있음을 확인하였다. As shown in FIG. 5 , it was confirmed that the CT-V2-derived stem cells according to the present invention had superior adipocyte differentiation ability compared to IVS and CT-V1-derived stem cells.
실시예 6: 탈락막 인접 융모간강(CT-V2) 유래된 줄기세포의 연골세포 분화능 확인Example 6: Confirmation of chondrocyte differentiation ability of stem cells derived from decidua adjacent interchorionic space (CT-V2)
상기 실시예 1에서 수득한 탈락막 인접 융모간강(CT-V2)으로부터 유래된 줄기세포의 연골세포로의 분화능을 확인하기 위하여, 줄기세포를 공지된 연골세포 분화 유도 배지(0.1μM 덱사메타손, 50㎍/㎖ 아스코르브산, 40㎍/㎖ L-프롤린, 10ng/㎖ TGF-β500ng/㎖ BMP-6, 50mg/ml ITS premix가 포함된 DMEM 배지)에서 3주 동안 배양하여 연골세포로의 분화를 유도하였다. 상기 줄기세포의 연골세포로의 분화 정도를 측정하기 위하여, 종래 공지된 방법에 따라 Safranin-O 염색 및 Type II 콜라겐을 이용한 면역화학염색법을 수행하였다. 또한, 3주 동안 배양 후, Glycan sulfated GAG assay kit (Biocolor Ltd., Carrickfergus, County Antrim, UK)를 사용하여 Sulfated glycosaminoglycan (GAG) 함량을 측정 하였다. 흡광도는 마이크로 플레이트 판독기를 사용하여 파장 656 nm에서 96-웰 플레이트에서 측정 하였고 결과를 도 6 에 나타내었다. In order to confirm the differentiation ability of stem cells derived from the decidual interchorionic space (CT-V2) obtained in Example 1 into chondrocytes, the stem cells were subjected to a known chondrocyte differentiation induction medium (0.1 μM dexamethasone, 50 μg). /ml ascorbic acid, 40㎍/ml L-proline, 10ng/ml TGF-β500ng/ml BMP-6, 50mg/ml ITS premix (DMEM medium containing premix) for 3 weeks to induce differentiation into chondrocytes . In order to measure the degree of differentiation of the stem cells into chondrocytes, Safranin-O staining and immunochemical staining using Type II collagen were performed according to a conventionally known method. In addition, after culturing for 3 weeks, the content of sulfated glycosaminoglycan (GAG) was measured using a glycan sulfated GAG assay kit (Biocolor Ltd., Carrickfergus, County Antrim, UK). Absorbance was measured in a 96-well plate at a wavelength of 656 nm using a microplate reader, and the results are shown in FIG. 6 .
도 6에 나타낸 바와 같이, 본 발명에 따른 CT-V2 유래 줄기세포는 IVS 와 CT-V1 유래 줄기세포에 비해 우수한 연골세포 분화능을 가지고 있음을 확인하였다.As shown in FIG. 6 , it was confirmed that the CT-V2-derived stem cells according to the present invention had superior chondrocyte differentiation ability compared to IVS and CT-V1-derived stem cells.
실시예 7: 탈락막 인접 융모간강(CV-V2) 유래 줄기세포의 골세포로의 분화능 확인Example 7: Confirmation of differentiation ability of stem cells derived from decidua adjacent interchorionic space (CV-V2) into osteocytes
상기 실시예 1에서 수득한 탈락막 인접 융모간강(CT-V2)에서 수득한 줄기세포의 골세포로의 분화능을 확인하기 위하여, 줄기세포를 공지된 골세포 분화 유도 배지(10% FBS, 1% anti-biotics, 100μM 덱사메타손, 50mM 아스코르브산-2-포스페이트, 10μM β글리크로포스페이트, 250μM 아스코르브산이 포함된 DMEM 배지)에서 4주 동안 배양하여 골세포로의 분화를 유도하였다. 이때, 분화유도 시작 후 2주가 경과한 시점에서는 종래 공지된 방법에 따라 ALP(Alkaline phosphate) 염색법으로 염색하였고, 4주가 경과한 시점에서는 종래 공지된 방법에 따라 Alizarin red S 염색법으로 염색함으로써, 골세포로의 분화 정도를 분석하였다. 또한, 동일한 방법으로 상기 비교예 1에서 수득한 태반 전체 및 다른 태반 세부조직 유래 줄기세포의 골세포로의 분화능을 4주가 경과한 시점에서 세포내 칼슘 측정을 하기 위해 24시간동안 0.6M HCl 을 처리하여 37℃에서 보관 후, o-cresolphthalein complexone method (Pointe Scientific, Canton, MI, USA )를 이용하여 565nm 에서 흡광도를 측정 후 칼슘농도를 표준화하고 정량화하였다. 그 결과를 도 7에 나타내었다. In order to confirm the differentiation ability of the stem cells obtained from the interchorionic space (CT-V2) adjacent to the decidua obtained in Example 1 into osteocytes, the stem cells were subjected to a known osteocytic differentiation induction medium (10% FBS, 1% Anti-biotics, 100 μM dexamethasone, 50 mM ascorbic acid-2-phosphate, 10 μM β-glycrophosphate, and DMEM medium containing 250 μM ascorbic acid) were cultured for 4 weeks to induce differentiation into osteocytes. At this time, when 2 weeks have elapsed from the start of differentiation induction, the cells were stained with Alkaline phosphate (ALP) staining according to a conventionally known method. The degree of differentiation into furnaces was analyzed. In addition, in the same manner, 0.6M HCl was treated for 24 hours to measure intracellular calcium at 4 weeks after the differentiation ability of the whole placenta and other placental subtissue-derived stem cells obtained in Comparative Example 1 into osteocytes in the same manner. After storage at 37°C, absorbance was measured at 565 nm using the o-cresolphthalein complexone method (Pointe Scientific, Canton, MI, USA), and calcium concentration was standardized and quantified. The results are shown in FIG. 7 .
도 7에 나타낸 바와 같이, 본 발명에 따른 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 IVS 와 CT-V1 에서 분리된 줄기세포에 비해 골세포로 분화될 수 있는 우수한 골세포 분화능을 가지고 있음을 확인하였다.As shown in FIG. 7 , the stem cells derived from the decidual interchorionic space (CT-V2) according to the present invention have superior osteocytic differentiation ability that can be differentiated into osteocytes compared to the stem cells isolated from IVS and CT-V1. confirmed that there is.
실시예 8: 탈락막 인접 융모간강(CT-V2) 유래 줄기세포의 세포 치료제 효과 검증 (연골 결손 동물모델) Example 8: Verification of the cell therapeutic effect of stem cells derived from the decidua adjacent chorionic space (CT-V2) (cartilage defect animal model)
상기 실시예 1에서 수득한 탈락막 인접 융모(CT-V2) 로부터 유래된 줄기세포의 연골 결손 동물모델에서 세포 치료제로서의 효과를 검증하기 위해, 하기와 같은 실험을 수행하였다. 보다 구체적으로, 18주령 래트(rat) 관절 연골 손상 동물모델을 제작하기 위하여, 건강한 래트를 선택하여 체중에 따른 적절한 양의 케타민과 럼푼으로 주사마취 한 후, 래트가 충분히 전신마취가 되었음을 확인하고, 양쪽 하지의 슬관절 부위를 면도한 후, 자세를 유지시키면서 반창고로 고정하였다. 양측 슬관절 부위를 포비돈으로 소독하고, 슬개골을 촉지하여 위치를 확인한 후, 슬관절의 위, 아래, 슬개골의 내측을 지나는 절개선을 따라 방정중 접근(paramedian approach)으로 슬관절 내에 도달하고, 슬개골을 외측으로 젖히면서 슬관절을 굴곡시켜 관절 내부를 관찰하였다. 특이한 병적 소견이 없음을 확인한 후, 슬개골구 중앙 과간와(顆間窩, interchondylar notch)의 상단 앞 끝에서 1㎜ 위쪽에 뾰족한 송곳으로 흠집을 낸 후, 이를 중심으로 하여 드릴로 지름 2㎜, 깊이 3㎜의 구멍을 만들어 전층(full thickness)에 손상을 주었다. 상기와 같이 연골 손상을 유발한지 16주 후에 손상 부위를 관찰하여, 연골 손상 부위가 천연 치유되지 않음을 확인하였다.In order to verify the effect of stem cells derived from the decidual adjacent villi (CT-V2) obtained in Example 1 as a cell therapeutic agent in an animal model of cartilage defect, the following experiment was performed. More specifically, in order to produce an 18-week-old rat articular cartilage damage animal model, a healthy rat was selected and anesthetized with appropriate amounts of ketamine and rumpun according to body weight, and then it was confirmed that the rat was sufficiently under general anesthesia, After shaving the knee joint of both lower extremities, it was fixed with a band-aid while maintaining the posture. Disinfect both knee joints with povidone, palpate the patella to confirm the position, and follow the incision line passing above, below and inside the patella to reach the inside of the knee joint with a paramedian approach, and then move the patella outward. The inside of the joint was observed by flexing the knee joint while bending it. After confirming that there are no specific pathological findings, make a scratch with a sharp awl 1 mm above the upper front end of the central interchondylar notch of the patellar fossa. A 3 mm hole was made and the full thickness was damaged. As described above, the damaged site was observed 16 weeks after induction of cartilage damage, and it was confirmed that the cartilage damaged site did not heal naturally.
주사기를 이용하여 4% 의 히알루론산과 본 발명에 따른 5 Х 106 cells/ml 의 CT-V2 또는 CT-V1 유래 줄기세포를 섞은 뒤, 500㎕를 상기 동물 모델의 오른쪽에 만들어진 연골 손상 부위에 주입하였다. 이후 슬개골을 원래 위치로 되돌린 후, 슬개골 주위의 연부 조직을 흡수성 실로 봉합하고, 피부를 비흡수성실로 봉합하였다. 반대측 다리에는 양성대조군으로 히알루론산 나트륨을 동량으로 주입하였다. 래트가 마취에서 깨어나는 것을 확인한 후 자유롭게 움직일 수 있도록 허용하였으며, 수술 후 5일간 감염을 막기 위해 진통제와 항생제를 투여하였다. 16주가 경과한 후, 각 래트로부터 손상 및 치료를 수행하였던 관절 연골 부위를 얻어 육안 및 ICRS(International Cartilage Repair Society) macroscopic score 평가를 수행하였다. 또한, 이들 조직으로부터 파라핀 블록을 제작하여 H&E, Safranin O 및 type II collagen 염색을 수행하였으며, Modified O'driscoll score를 이용한 정량화를 통해 재생된 조직을 분석하였다. 그 결과를 도 8, 도9, 도10 및 도 11에 각각 나타내었다.After mixing 4% hyaluronic acid and 5 Х 10 6 cells/ml of CT-V2 or CT-V1-derived stem cells according to the present invention using a syringe, 500 μl of the cartilage damage site created on the right side of the animal model injected. After returning the patella to its original position, the soft tissue around the patella was sutured with an absorbable thread, and the skin was sutured with a non-absorbable thread. An equal amount of sodium hyaluronate was injected into the contralateral leg as a positive control. After confirming that the rats woke up from anesthesia, they were allowed to move freely, and analgesics and antibiotics were administered to prevent infection for 5 days after surgery. After 16 weeks had elapsed, the joint cartilage area that had been damaged and treated was obtained from each rat, and macroscopic and ICRS (International Cartilage Repair Society) macroscopic score evaluation was performed. In addition, paraffin blocks were prepared from these tissues and stained with H&E, Safranin O and type II collagen, and the regenerated tissues were analyzed through quantification using the Modified O'driscoll score. The results are shown in FIGS. 8, 9, 10 and 11, respectively.
도 8에 나타낸 바와 같이, CT-V2 또는 CT-V1 유래 줄기세포 및 히알루론산 나트륨을 투여하고 16주 후에 육안으로 관절 병변 부위를 확인한 결과, 줄기세포 실험군에서는 대조군에 비해 연골 표면이 더 매끄럽고 기존 조직과의 유합도 잘되는 것을 확인하였다. As shown in FIG. 8 , after administration of CT-V2 or CT-V1-derived stem cells and sodium hyaluronate, the joint lesion site was visually confirmed 16 weeks later, in the stem cell experimental group, the cartilage surface was smoother than in the control group, and the existing tissue It was also confirmed that the union with
도 9에 나타낸 바와 같이, CT-V2 또는 CT-V1 유래 줄기세포를 투여하고 16주 후에 ICRSmacroscopic score 를 이용하여 평가한 결과, 대조군 (4.63±0.58), CT-V1 유래 줄기세포 (7.21±1.10), CT-V2 유래 줄기세포 (9.45±1.18) 순으로 높은 score 를 보였다. As shown in FIG. 9 , after 16 weeks of administration of CT-V2 or CT-V1-derived stem cells, the results were evaluated using ICRS macroscopic score, control group (4.63±0.58), CT-V1-derived stem cells (7.21±1.10) , CT-V2-derived stem cells (9.45±1.18) showed the highest score in that order.
도 10에 나타낸 바와 같이, CT-V2 또는 CT-V1 유래 줄기세포를 투여하고 16주 후에 H&E 염색 결과, 대조군의 경우, 결함 부위가 주변의 정상 연골 조직에 비해 함몰된 표면으로 불규칙한 것으로 나타났으며, 결손부위의 표면에 균열이나 균열이 관찰되었으며 주변 정상연골과의 밀접한 유합은 관찰되지 않았다. 반면, 줄기세포와 HA를 이식한 군에서는 새로 형성된 조직과 정상 관절 연골이 잘 결합되어 표층 및 심층에서 보다 성숙하고 완전한 세포 배열이 생성되었다. 또한 정상적인 연골 조직과 유사한 형태도 관찰되었다.As shown in FIG. 10 , as a result of H&E staining 16 weeks after administration of CT-V2 or CT-V1-derived stem cells, in the case of the control group, the defect site was found to be irregular with a recessed surface compared to the surrounding normal cartilage tissue. , cracks or cracks were observed on the surface of the defect site, and close union with surrounding normal cartilage was not observed. On the other hand, in the group transplanted with stem cells and HA, the newly formed tissue and normal articular cartilage were well combined, resulting in a more mature and complete cell arrangement in the superficial and deep layers. A morphology similar to that of normal cartilage was also observed.
Safranin O 염색 결과, 재생된 조직에서 형성되는 proteoglycan의 분포를 확인하였으며, 이는 대조군에 비해 MSC 처리군에서 우월한 것으로 관찰되었다. As a result of Safranin O staining, the distribution of proteoglycan formed in the regenerated tissue was confirmed, which was observed to be superior in the MSC-treated group compared to the control group.
type II collagen 염색 결과, 조직 재생 부위에 고르게 분포되어 있음을 확인하였다. As a result of type II collagen staining, it was confirmed that it was evenly distributed in the tissue regeneration site.
또한 도 11에 나타낸 바와 같이, Modified O'driscoll score 결과에서는 16주 후 대조군 (5.12±2.14), CT-V1 줄기세포 (15.56±2.96), CT-V2 줄기세포 (20.12±2.54) 순으로 높은 score 를 보였다. Also, as shown in FIG. 11 , in the Modified O'driscoll score result, after 16 weeks, the control group (5.12±2.14), CT-V1 stem cells (15.56±2.96), CT-V2 stem cells (20.12±2.54) had the highest score in that order. showed
따라서 CT-V2 줄기세포는 손상된 연골을 재생시켜 주는 역할을 하는 것으로 나타나는 것을 알 수 있었다. Therefore, it was found that CT-V2 stem cells appear to play a role in regenerating damaged cartilage.
이상의 실험 결과를 통하여, 종래의 태반 전체로부터 유래한 줄기세포가 다양한 특성을 가지는 세부조직으로부터 유래된 줄기세포가 혼합되어 있어 서로 다른 세포로 분화하는 능력이 균일하게 나타나지 않는데 반해, 본 발명에 따른 태반의 세부조직인 탈락막 인접 융모간강(CT-V2) 유래 줄기세포는 우수한 분화능과 기타 줄기세포의 여러 특성에 대한 균질성의 측면에서 볼 때, 종래의 태반 전체 유래 줄기세포에 비하여 우수한 특성을 나타냄을 확인할 수 있었다. 특히 본 발명에 따른 CT-V2 유래 줄기세포는 다른 태반 세부조직 유래 줄기세포보다 성장, 증식, 형태 및 분화의 특성에서 일관된 양상을 보여 주었고, 가장 우수한 줄기세포의 특성을 보여주었다. 따라서 상기 CT-V2 유래 줄기세포를 이용할 경우 목적하는 세포로의 분화 효율을 향상시킬 수 있으며, 다양한 질환에서 세포치료제로 유용하게 이용할 수 있음을 확인하였다. Through the above experimental results, the conventional stem cells derived from the whole placenta are mixed with stem cells derived from detailed tissues having various characteristics, so the ability to differentiate into different cells does not appear uniformly, whereas the placenta according to the present invention is not uniformly displayed. It was confirmed that stem cells derived from the decidua adjacent interchorionic space (CT-V2), a detailed tissue of could In particular, the CT-V2-derived stem cells according to the present invention showed consistent aspects in the characteristics of growth, proliferation, morphology and differentiation than other placental tissue-derived stem cells, and showed the best stem cell characteristics. Therefore, it was confirmed that, when the CT-V2-derived stem cells are used, the differentiation efficiency into a target cell can be improved, and it can be usefully used as a cell therapy agent in various diseases.
Claims (19)
- 탈락막 인접 융모간강 유래 줄기세포로, 상기 탈락막 인접 융모간강은 전체 영양막 층 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인, 탈락막 인접 융모간강 유래 줄기세포.Decidual proximal interchorionic space-derived stem cells, wherein the decidual adjacent interchorionic space is a trophoblast from the decidua proximal site to 2/3 of the entire trophoblast layer, the decidual proximal interchorionic space-derived stem cells.
- 제1항에 있어서, 상기 줄기세포는 CD24, CD104, CD227, Disialogangolioside GD2 또는 SSEA-4 에 대하여 음성의 표면인자 발현 특성을 갖는 것을 특징으로 하는, 줄기세포. According to claim 1, wherein the stem cells are CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4 characterized in that it has a negative surface factor expression characteristics for, stem cells.
- 제1항에 있어서, 상기 줄기세포는 CD49b, CD74 또는 CD200에 대하여 양성의 표면인자 발현 특성을 갖는 것을 특징으로 하는, 줄기세포. According to claim 1, wherein the stem cells are CD49b, CD74, or CD200, characterized in that it has a positive surface factor expression characteristics, stem cells.
- 제1항에 있어서, 상기 줄기세포는 CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 또는 SSEA-4에 대하여 음성의 표면인자 발현; 및 CD44, CD73, CD90, CD105, CD49b, CD74 또는 CD200 에 대하여 양성의 표면인자 발현 특성을 갖는 것을 특징으로 하는, 줄기세포. According to claim 1, wherein the stem cells are CD31, CD34, Cd45, HLA-DR, CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4 negative surface factor expression; and CD44, CD73, CD90, CD105, CD49b, CD74 or CD200, characterized in that it has a positive surface factor expression characteristic, stem cells.
- 제1항에 있어서, 상기 줄기세포는 모체 세포 기원의 줄기세포인, 줄기세포. According to claim 1, wherein the stem cells are stem cells of parental cell origin, stem cells.
- 제1항에 있어서, 상기 줄기세포는 The method of claim 1, wherein the stem cells are1) 전체 영양막층 중 탈락막 인접 부위에서부터 2/3 지점까지인 영양막층 부위를 절단하여 탈락막 인접 영양막층을 얻는 단계; 1) obtaining a trophoblast adjacent to the decidua by cutting the portion of the trophoblast that is from the portion adjacent to the decidua to the 2/3 point among the entire trophoblast layer;2) 상기 탈락막 인접 영양막층에서 융모를 제거하여 탈락막 인접 융모간강을 얻는 단계; 및2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and3) 상기 탈락막 인접 융모간강에서 줄기세포를 수득하는 단계; 를 통해 수득되는 것을 특징으로 하는, 줄기세포. 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; Characterized in that obtained through, stem cells.
- 1) 전체 영양막층 중 탈락막 인접 부위에서부터 2/3 지점까지인 영양막층 부위를 절단하여 탈락막 인접 영양막층을 얻는 단계; 1) obtaining a trophoblast adjacent to the decidua by cutting the portion of the trophoblast that is from the portion adjacent to the decidua to the 2/3 point among the entire trophoblast layer;2) 상기 탈락막 인접 영양막층에서 융모를 제거하여 탈락막 인접 융모간강을 얻는 단계; 및2) removing the villi from the trophoblast layer adjacent to the decidua to obtain an interchorionic cavity adjacent to the decidua; and3) 상기 탈락막 인접 융모간강에서 줄기세포를 수득하는 단계; 를 포함하는 탈락막 인접 융모간강 유래 줄기세포의 분리 수득 방법. 3) obtaining stem cells from the interchorionic cavity adjacent to the decidua; A method for separating and obtaining stem cells derived from the interchorionic cavity adjacent to the decidua comprising a.
- 제7항에 있어서, 상기 탈락막 인접 융모간강 유래 줄기세포는 CD24, CD104, CD227, Disialogangolioside GD2 또는 SSEA-4 에 대하여 음성의 표면인자 발현 특성을 갖는 것을 특징으로 하는, 방법. The method of claim 7, wherein the decidual adjacent interchorionic stem cells have negative surface factor expression characteristics for CD24, CD104, CD227, Disialogangolioside GD2 or SSEA-4.
- 제7항에 있어서, 상기 줄기세포는 CD49b, CD74 또는 CD200에 대하여 양성의 표면인자 발현 특성을 갖는 것을 특징으로 하는, 방법. The method of claim 7, wherein the stem cells have positive surface factor expression characteristics for CD49b, CD74 or CD200.
- 제1항의 줄기세포를 유효성분으로 포함하는 조직 재생용 세포치료제.A cell therapy product for tissue regeneration comprising the stem cell of claim 1 as an active ingredient.
- 제10항에 있어서, 상기 조직은 지방, 연골, 골, 신경, 인대 및 건으로 이루어진 군에서 선택된 1종 이상인, 조직 재생용 세포 치료제. The cell therapeutic agent for tissue regeneration according to claim 10, wherein the tissue is at least one selected from the group consisting of fat, cartilage, bone, nerve, ligament, and tendon.
- 제11항에 있어서, 상기 연골은 유리연골, 섬유연골 또는 탄성연골인 것을 특징으로 하는, 조직 재생용 세포 치료제. The cell therapeutic agent for tissue regeneration according to claim 11, wherein the cartilage is free cartilage, fibrocartilage or elastic cartilage.
- 제11항에 있어서, 상기 연골은 관절연골(articular Cartilage), 귀 연골, 비연골, 팔꿈치 연골, 반월상연골(meniscus), 무릎연골, 늑연골, 발목연골, 관연골, 후두연골 및 척추 연골로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 조직 재생용 세포 치료제. According to claim 11, wherein the cartilage is articular cartilage (articular cartilage), ear cartilage, nasal cartilage, elbow cartilage, meniscus (meniscus), knee cartilage, cost cartilage, ankle cartilage, ductal cartilage, occipital cartilage and vertebral cartilage consisting of A cell therapeutic agent for tissue regeneration, characterized in that it is selected from the group.
- 제11항에 있어서, 상기 세포치료제는 골 결손, 건-인대 결손, 지방조직 결손, 연골 괴사, 골연골염, 연골파열, 연골외상, 관절염, 연골결핍 또는 선천성 기관연화 치료용인 것을 특징으로 하는, 조직 재생용 세포치료제.The tissue of claim 11, wherein the cell therapy agent is for treatment of bone defect, tendon-ligament defect, adipose tissue defect, cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma, arthritis, cartilage deficiency or congenital organ softening. Cell therapy for regeneration.
- 제10항에 있어서, 상기 조직재생용 세포 치료제는 주사제인 것을 특징으로 하는, 조직 재생용 세포 치료제. The cell therapy for tissue regeneration according to claim 10, wherein the cell therapy for tissue regeneration is an injection.
- 제15항에 있어서, 상기 조직 재생용 세포 치료제는 하이드로겔을 더 포함하는 것을 특징으로 하는, 조직 재생용 세포 치료제.The cell therapy for tissue regeneration according to claim 15, wherein the cell therapy for tissue regeneration further comprises a hydrogel.
- 제16항에 있어서, 상기 하이드로겔은 소장점막하 조직, 히알루론산(hyalurinic acid), 카복시메틸셀룰로오스(CMC, carboxymethylcellulose), 알지네이트(alginate), 키토산(chitosan), 폴리아크릴아미드(polyacrylamide), 폴리(N-이소프로필아크릴아미드)(poly(N-isopropylacrylamide)), β글리세로포스페이트(β플루로닉(Poly(ethylene oxide)poly(propylene oxide)poly(ethyleneoxide), Pluronic), 피브린, 폴리에틸렌옥사이드 및 카복시메틸셀룰로오스(CMC)와 폴리에틸렌이민(PEI)의 혼합물로 이루어진 군에서 선택된 1종 이상으로 이루어진 군에서 선택되는 것을 특징으로 하는, 조직 재생용 세포 치료제.The method of claim 16, wherein the hydrogel is small intestine submucosal tissue, hyaluronic acid (hyalurinic acid), carboxymethyl cellulose (CMC, carboxymethylcellulose), alginate (alginate), chitosan (chitosan), polyacrylamide (polyacrylamide), poly (N) -isopropylacrylamide) (poly(N-isopropylacrylamide)), β glycerophosphate (β pluronic (Poly(ethylene oxide)poly(propylene oxide)poly(ethyleneoxide), Pluronic), fibrin, polyethylene oxide and carboxymethyl Cell therapy for tissue regeneration, characterized in that it is selected from the group consisting of at least one selected from the group consisting of a mixture of cellulose (CMC) and polyethyleneimine (PEI).
- 제1항의 줄기세포를 유효성분으로 포함하는 조직 재생용 조성물.A composition for tissue regeneration comprising the stem cell of claim 1 as an active ingredient.
- 탈락막 인접 융모간강 유래 줄기세포를 이를 필요로 하는 개체에 처리하는 단계; 를 포함하는 조직재생 방법으로, treating the decidual adjacent interchorionic space-derived stem cells to an individual in need thereof; As a tissue regeneration method comprising a,상기 탈락막 인접 융모간강은 전체 영양막층 중 탈락막 인접 부위에서부터 2/3 지점까지 부위의 영양막인, 조직 재생방법. The decidual adjacent interchorionic cavity is a trophoblast of the area from the decidua proximal site to the 2/3 point of the entire trophoblast layer, a tissue regeneration method.
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