Chorionic plate adjacent villus derived stem cells and cell therapy agent for tissue regeneration containing the same

Abstract

The present invention relates to stem cells derived from villi adjacent to chorionic plate and cell therapeutic agents containing the same. The villi adjacent to chorionic plate (VCP) tissue-derived stem cells of the present invention are characterized in that the villi adjacent to chorionic plate tissue is located in the region from the chorionic plate adjacent part of the whole villus to 1 / 3 of the distance from the chorionic plate adjacent part to the end of the villus, including the villus base part. Compared with other placental tissue-derived stem cells, they exhibit uniform growth characteristics, excellent proliferation characteristics, and significantly superior differentiation ability to cartilage, bone, and fat. Therefore, they can be effectively used in various tissue regeneration treatments required for cartilage, bone, and fat regeneration, and in particular, can be effectively used in the treatment of cartilage regeneration and osteoarthritis.

Description

Technical Field

[0001] The present invention relates to stem cells derived from the villi adjacent to the chorionic plate and a cell therapeutic agent containing the same. Background Art

[0002] Stem cells are cells that have the ability to self-replicate and differentiate into more than two cells. They can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells. Totipotent stem cells are pluripotent cells that can develop into a complete individual. After the egg and sperm are fertilized, the cells until the 8-cell stage have this property. If these cells are separated and transplanted into the uterus, they can develop into a complete individual. Pluripotent stem cells are cells that can develop into tissues with various cells derived from the ectoderm, mesoderm, and endoderm. They are derived from the inner cell mass in the blastocyst that appears 4 to 5 days after fertilization. They are called embryonic stem cells. Although they can differentiate into various different tissue cells, they cannot form new life. Multipotent stem cells are stem cells that can only differentiate into specific cells in the tissues and organs containing these cells. It is known that pluripotent stem cells as described above are derived from adult bone marrow, skin, blood vessels, muscles, etc., and such stem cells are rapidly being applied to the fields of tissue engineering, gene therapy, and cell therapy agents. In addition, there is an urgent need to obtain stem cells from multiple tissues and use them in various applications.

[0003] When a woman is pregnant, the placenta, an organ that develops from the uterine wall, shows a disc-shaped shape with rich vascular tissue. The fetus's nutrition intake, breathing, and excretion are all achieved through the placenta. According to scientific research results, lifelong health depends on the fetal period. Therefore, the importance of the placenta during pregnancy is being emphasized, and attempts are being made to understand the relationship between various substances related to this. Although the placenta is composed of various forms of cells based on different weeks and positions, its scope of application is still very limited.

[0004] Recently, research is being conducted on stem cells isolated from placental tissue. Most of the existing placental tissue-derived stem cells are stem cells derived from partial tissues of the full-term placenta that can be obtained during childbirth. The placenta is composed of a variety of tissues, including the basement membrane attached to the uterus, the chorionic tissue that mediates the exchange of substances between the maternal and fetal blood as the central tissue of the placenta, and the chorionic membrane that covers the placenta along the inside of the placenta and surrounds the fetus and amnion, but so far, research on its local tissues has not been widely known. On the other hand, Korean Patent No. 818214 discloses a method for isolating stem cells from the amnion or decidua using a culture medium containing N-acetyl-L-cysteine ​​(NAC), and Korean Patent No. 871984 discloses the pluripotency of stem cells derived from the amnion, serosa, basal decidua and placental tissue using a culture medium containing basic fibroblast growth factor (bFGF, Basic Fibroblast Growth Factor).

[0005] However, to date, there has been no extensive report on the placenta division method that is not limited to the placental structure division method currently known in anatomy and the stem cells obtained from the division site and their effects. Summary of the invention

[0006] Technical issues

[0007] In this regard, in the process of redefining the local area of ​​the placenta without being limited to the existing placental tissue separation method and studying the characteristics of stem cells based on this, the inventors obtained the villi adjacent to the chorionic plate (VCP) and compared the stem cells isolated therefrom with the whole placenta, the whole villi and the villus-derived stem cells in the remaining part of the whole villi except the villi adjacent to the chorionic plate to confirm the different CD marker expression patterns and the excellent differentiation ability into cartilage, bone and fat, thereby completing the present invention.

[0008] Therefore, the object of the present invention is to provide stem cells derived from chorionic plate adjacent villus tissue, wherein the chorionic plate adjacent villus is located in the chorionic plate adjacent part of the whole villus to 1 / 3 of the distance from the chorionic plate adjacent part to the villus end, including the villus base part.

[0009] Furthermore, another object of the present invention is to provide a cell therapy agent comprising the above-mentioned chorionic plate adjacent villus tissue-derived stem cells, a composition for tissue regeneration, and a method for isolating the same.

[0010] Technical Solution

[0011] In order to achieve the above object, the present invention provides stem cells derived from chorionic plate adjacent villus tissue, wherein the chorionic plate adjacent villus is the area from the chorionic plate adjacent part in the whole villus to 30% of the length of the chorionic plate adjacent part in the whole villus, including the villus base part.

[0012] In addition, the present invention provides a method for isolating and obtaining stem cells derived from chorionic plate adjacent villi, which comprises the following steps: 1) cutting the chorionic plate adjacent portion from the whole villi to the region from the chorionic plate adjacent portion to 1 / 3 of the entire villus length to obtain chorionic plate adjacent villi; and 2) obtaining stem cells from the chorionic plate adjacent villi.

[0013] Furthermore, the present invention provides a method for preparing stem cells derived from villi adjacent to the chorionic plate, which comprises the following steps: 1) cutting the chorionic plate adjacent portion from the whole villus to the region from the chorionic plate adjacent portion to 1 / 3 of the entire villus length to obtain villi adjacent to the chorionic plate; and 2) obtaining stem cells from the villi adjacent to the chorionic plate.

[0014] Furthermore, the present invention provides a cell therapy agent for tissue regeneration comprising the above-mentioned stem cells as an active ingredient.

[0015] Furthermore, the present invention provides a composition for tissue regeneration comprising the above-mentioned stem cells as an effective ingredient.

[0016] Furthermore, the present invention provides a tissue regeneration method, which includes the step of treating an individual in need thereof using chorionic plate adjacent villus tissue-derived stem cells derived from chorionic plate adjacent villus tissue, wherein the chorionic plate adjacent villus tissue is located in the area from the chorionic plate adjacent part to 1 / 3 of the distance from the chorionic plate adjacent part to the end of the villus in the whole villus, including the villus base part.

[0017] Furthermore, the present invention provides a tissue regeneration method, which comprises the following steps: 1) cutting the area from the chorionic plate adjacent portion to 1 / 3 of the entire villus length from the chorionic plate adjacent portion to obtain villi adjacent to the chorionic plate; 2) obtaining stem cells from the villi adjacent to the chorionic plate; and 3) using the obtained villus-derived stem cells adjacent to the chorionic plate to treat an individual in need thereof.

[0018] Effects of the Invention

[0019] The chorionic plate adjacent villus tissue-derived stem cells of the present invention are characterized in that the chorionic plate adjacent villus tissue is located in the area from the chorionic plate adjacent site to 1 / 3 of the distance from the chorionic plate adjacent site to the end of the villus in the whole villus, including the villus basal site. Compared with other embryonic tissue-derived stem cells, they exhibit uniform growth characteristics, excellent proliferation characteristics, and significantly superior differentiation ability to cartilage, bone, and fat. Therefore, they can be effectively used in various tissue regeneration treatments required for cartilage, bone, and fat regeneration, and in particular, can be effectively used to treat cartilage regeneration and osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A diagram showing the chorionic plate, the entire villus (CV), and the villi adjacent to the chorionic plate (VCP).

[0021] Figure 2 The figure shows the results of observing cell morphology under a microscope after culturing whole placenta-derived stem cells (Pla), whole villus-derived stem cells (CV), chorionic plate adjacent villus (VCP)-derived stem cells (CV1), and villus-derived stem cells in whole villi excluding chorionic plate adjacent villi (CV-VCP) (CV2).

[0022] Figure 3 Part A is a graph showing the results of confirming the colony-forming ability of whole placenta-derived stem cells (Pla), whole chorionic villus-derived stem cells (CV), chorionic plate adjacent villus-derived stem cells (CV1), and stem cells derived from sites in the whole villus excluding chorionic plate adjacent villi (CV-VCP) (CV2). Figure 3 Part B is a graph showing the colony size of CV1.

[0023] Figure 4 The figure shows the results of comparing the population doubling time of whole placenta-derived stem cells (Pla), whole chorionic villus-derived stem cells (CV), chorionic plate adjacent villus (VCP)-derived stem cells (CV1), and stem cells derived from sites in the whole villus excluding chorionic plate adjacent villus (CV-VCP) (CV2) according to P2 to P6 subculture.

[0024] Figure 5 The figure shows the results of confirming the chondrogenic differentiation ability of whole placenta-derived stem cells (Pla), whole villus-derived stem cells (CV), chorionic plate adjacent villus (VCP)-derived stem cells (CV1), and stem cells derived from sites in whole villi excluding chorionic plate adjacent villi (CV-VCP). Figure 5 Part A is a diagram showing the results of saponin O staining. Figure 5 Part B is a diagram showing the results of type II collagen staining. Figure 5 Part C is a graph showing the results of GAG content measurement.

[0025] Figure 6 The figure shows the results of confirming the osteoblast differentiation ability of whole placenta-derived stem cells, whole chorionic villus-derived stem cells (CV), chorionic plate adjacent villus-derived stem cells (CV1), and stem cells derived from the site of whole villus excluding chorionic plate adjacent villus (CV-VCP) (CV2). Figure 6 Part A is a graph showing the results of alkaline phosphatase (ALP) staining. Figure 6 Part B is a diagram showing the results of Alizarin red S staining. Figure 6 Part C is a graph showing the results of calcium content determination.

[0026] Figure 7 The figure shows the results of confirming the adipocyte differentiation ability of whole placenta-derived stem cells (Pla), whole villus-derived stem cells (CV), chorionic plate adjacent villus (VCP)-derived stem cells (CV1), and stem cells derived from the area of ​​whole villus excluding chorionic plate adjacent villus (CV-VCP) (CV2). Figure 7 Part A of FIG. 1 is a diagram showing the results of Oil red O staining. Figure 7 Part B is a graph quantitatively showing the results of Oil Red O staining.

[0027] Figure 8 This is a graph showing the results of examining the extent of cartilage regeneration after administration of CV1 to a cartilage damage animal model.

[0028] Fig. 9 This figure shows the results of measuring the OARSI score after administering CV1 to a cartilage damage animal model to confirm the cartilage protection and regeneration effects during the progression of arthritis. DETAILED DESCRIPTION

[0029] The present invention provides chorionic plate adjacent villus tissue derived stem cells and a cell therapeutic agent for tissue regeneration and a composition for tissue regeneration containing the same.

[0030] Hereinafter, the present invention will be described in detail.

[0031] In the present invention, "stem cells" refer to cells that have the ability to self-replicate and differentiate into two or more types of cells that are not mutually connected. According to their differentiation ability, stem cells can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.

[0032] In the present invention, "placenta" refers to the living tissue used to nurture the fetus during pregnancy, which is disc-shaped, has a weight of 500g to 600g, a diameter of 15cm to 20cm, and a thickness of 2cm to 3cm. One side of the placenta is in contact with the mother, and the other side is in contact with the fetus. In between, it is used to transport nutrients and oxygen between the mother's blood and the fetus's blood vessels. The placenta is roughly divided into three layers, namely, the amnion, the chorion, and the decidua. In more detail, it can be divided into the amniotic epithelium, the amnion, the chorion, the trophoblast, and the decidua.

[0033] The "chorionic plate adjacent villi" of the present invention includes the villus base part, the chorionic plate adjacent part in the whole villi to the 1 / 3 area of ​​the distance from the chorionic plate adjacent part to the villus end, more specifically, the continuous part starting from the chorionic plate adjacent part side corresponding to the 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3 area of ​​the distance from the chorionic plate adjacent part to the villus end, preferably, the chorionic plate adjacent part The position is from 1 / 5 to 1 / 3 of the distance from the part adjacent to the chorionic plate to the end of the villus, that is, in the whole villus, a continuous part starting from the part adjacent to the chorionic plate to 20% or 33% of the length of the whole villus. For example, it can be the area starting from the part adjacent to the chorionic plate to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the length of the whole villus. Figure 1 The chorionic plate of the present invention is shown adjacent to the villi.

[0034] The present invention provides stem cells derived from chorionic plate adjacent villus tissue, wherein the chorionic plate adjacent villus tissue derived stem cells are characterized in that the chorionic plate adjacent villus is a region from the chorionic plate adjacent part to 1 / 3 of the distance from the chorionic plate adjacent part to the end of the villus in the whole villus, including the villus base part.

[0035] The chorionic plate adjacent villi (VCP) tissue-derived stem cells of the present invention have negative surface factor expression characteristics for CD273, CD275 and / or CD321, which is an inherent feature different from the whole placenta-derived stem cells, the whole villus (CV)-derived stem cells and the remaining villi (CV-VCP)-derived stem cells in the whole villi except the villi adjacent to the chorionic plate defined in the present invention, which show positive surface factor expression characteristics for CD273, CD275 and CD321, and can show that the stem cells of the present invention are different from other stem cells.

[0036] Furthermore, the stem cells of the present invention may have positive surface factor expression characteristics for CD49e, CD140b and / or CD142.

[0037] The chorionic plate adjacent villus tissue-derived stem cells of the present invention may have the following surface factor marker expression characteristics: i) positive for CD44, CD73, CD90 and CD105; ii) positive for CD49e, CD140b or CD142; iii) negative for CD31, CD34, CD45 and HLA-DR; and iv) negative for CD273, CD275 or CD321. For example, the surface factor marker expression characteristics of being positive for CD44, CD73, CD90, CD105 and negative for CD31, CD34, CD45 and HLA-DR may be negative for one or more markers selected from the group consisting of CD49e, CD140b and CD142, and may be negative for one or more markers selected from the group consisting of CD273, CD275 and CD321. As an example, the surface factor marker expression characteristics are positive for CD49e, CD140b, and CD142, and negative for CD273, CD275, and CD321.

[0038] Furthermore, the stem cells of the present invention are characterized in that they can be stem cells derived from embryonic cells (cell origin). The placenta, as an intrinsic tissue that expresses fetal and maternal derived characteristics, is composed of a variety of tissues that express individual characteristics, including the basement membrane attached to the uterus, the chorionic tissue that mediates the exchange of substances between the maternal and fetal blood as the central tissue of the placenta, and the chorionic membrane that covers the placenta along the inside of the placenta and surrounds the fetus and the amniotic membrane. Therefore, even placenta-derived stem cells can show different cell sources according to the location of acquisition, for example, trophoblast-derived stem cells are derived from maternal cells, and chorionic-derived stem cells are derived from fetal cells. In the case of the entire placenta-derived stem cells, they are derived from the fetus and the mother. The results of chromosome type analysis show that the chorionic plate adjacent to the chorionic villus tissue-derived stem cells of the present invention are confirmed to have a fetal-derived cell chromosome type. This is because the remaining villus (CV-VCP) derived stem cells except the chorionic plate adjacent to the villi express maternal-derived chromosome types, so, according to the location of the divided acquisition site, even the same villus-derived stem cells have different cell sources and express different characteristics.

[0039] Furthermore, the stem cells of the present invention can be obtained by the following steps: 1) cutting the area adjacent to the chorionic plate from the whole villus to 1 / 3 of the distance from the area adjacent to the chorionic plate to the end of the villus to obtain the villi adjacent to the chorionic plate; and 2) obtaining stem cells from the villi adjacent to the chorionic plate.

[0040] Furthermore, the method for isolating and obtaining stem cells derived from chorionic plate adjacent villi of the present invention comprises the following steps: 1) obtaining chorionic plate adjacent villus tissue in the area from the chorionic plate adjacent site to 1 / 3 of the distance from the chorionic plate adjacent site to the end of the villus in the whole villus; and 2) obtaining stem cells from the chorionic plate adjacent villi.

[0041] Furthermore, in the present invention, the above-mentioned separation and acquisition method can be used in the same meaning as the method for preparing stem cells derived from chorionic plate adjacent villi. The present invention can provide a method for preparing stem cells derived from chorionic plate adjacent villi, which comprises: 1) obtaining chorionic plate adjacent villus tissue in the whole villus from the chorionic plate adjacent part to 1 / 3 of the distance from the chorionic plate adjacent part to the end of the villus; and 2) obtaining stem cells from the chorionic plate adjacent villi.

[0042] Step 1) of the above method is a step of obtaining the villi adjacent to the chorionic plate from the whole villi, and the villi adjacent to the chorionic plate can be obtained by using a known method in the art without limitation. In this case, the villi adjacent to the chorionic plate can be obtained by cutting the area from the whole villi to 1 / 3 of the distance from the chorionic plate adjacent area to the end of the villi using sterile forceps and a scalpel, and more specifically, the continuous area starting from the side of the chorionic plate adjacent area can be obtained by cutting the area corresponding to the 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, and 1 / 3 of the distance from the chorionic plate adjacent area to the end of the villi from the whole villi. Preferably, the continuous portion of 20% to 33% of the length of the whole villi is cut from the chorionic plate adjacent portion to the 1 / 5 region or the 1 / 3 region of the distance from the chorionic plate adjacent portion to the end of the villi, that is, the chorionic plate adjacent portion in the whole villi as the starting point, so that the whole villi can be divided into the chorionic plate adjacent villi and the remaining villi portion in the whole villi except the chorionic plate adjacent villi by such cutting. The chorionic plate adjacent villi include the villi base portion, and the CV-VCP portion includes the end portion of the villi.

[0043] The above step 2) is a step of obtaining stem cells from the adjacent villi of the chorionic plate obtained by step 1), and the cells obtained by adding enzyme solution to the adjacent villi tissue of the chorionic plate for enzyme reaction without using growth factors and cultured in a culture medium with added fetal bovine serum and antibiotics can be obtained by recovery. The above enzymes include trypsin, collagenase, dispase, deoxyribonuclease (DNase), ribonuclease (RNase), protease, lipase, hyaluronidase and elastase, but are not limited to this. The above collagenase includes collagenase A, collagenase I, collagenase II, collagenase III or collagenase IV, etc.

[0044] According to the method of the present invention, stem cells can also be specifically isolated from the chorionic plate adjacent villi in the villi. Different from whole villi, CV-VCP villi, and placenta-derived stem cells, the stem cells isolated by this method are characterized by having negative surface factor expression characteristics for CD273, CD275, and CD321. Compared with other tissue-derived stem cells, the stem cells obtained by the method of the present invention can show morphological characteristics of fibroblast shape, can specifically maintain only single cells, and can show excellent colony formation ability and proliferation ability.

[0045] The stem cells derived from the villous region adjacent to the chorionic plate of the present invention can be differentiated into various types of cells, for example, adipocytes, chondrocytes, osteocytes, nerve cells, ligament cells, or tenocytes, but are not limited thereto.

[0046] In the present invention, "differentiation" refers to the phenomenon that a relatively simple single line is divided into two or more sub-lineages with different properties. Specifically, it refers to the phenomenon that cells are specialized into different structures or functions in the process of cell division and proliferation. That is, it refers to the phenomenon that cells, tissues, etc. of organisms change their morphology or functions in order to perform their respective functions. In contrast, "undifferentiation" refers to a state where the above differentiation has not occurred and still has the characteristics of stem cells.

[0047] The differentiation method of stem cells can be performed by an existing known method, and there is no particular limitation on this. For example, the following methods can be preferably used, that is, a method of differentiating the above-mentioned stem cells into adipocytes by culturing them in a culture medium containing dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine (IBMX); a method of differentiating the above-mentioned stem cells into chondrocytes by culturing them in a culture medium containing dexamethasone, bone morphogenetic protein 6 (BMP-6), transforming growth factor β (TGF-β factor beta), ascorbic acid and L-proline; and a method of differentiating the above-mentioned stem cells into osteocytes by culturing them in a culture medium containing dexamethasone, ascorbic acid and β-glycerophosphate (β-ascorbic acid-2-phosphate trisodium salt).

[0048] The method for determining the degree of differentiation of stem cells derived from the chorionic plate adjacent to the villi differentiated by the above method is not limited thereto, and known methods in the art can be used, for example, flow cytometry, immunocytochemistry, methods of determining cell surface markers or morphological changes using polymerase chain reaction (PCR) or gene expression profiles, methods of examining cell morphological changes using an optical microscope or a confocal microscope, methods of determining changes in gene expression profiles, etc. Preferably, RT-PCR, Oil Red O staining, Safranin O staining, type II collagen immunohistochemistry staining, alkaline phosphate (ALP) staining, or Alizarin Red S staining, etc. can be used.

[0049] Furthermore, the present invention provides a cell therapy agent for tissue regeneration or a composition for tissue regeneration comprising stem cells derived from chorionic plate adjacent villus tissue as an effective ingredient, wherein the chorionic plate adjacent villus tissue is located in the area from the chorionic plate adjacent part to 1 / 3 of the distance from the chorionic plate adjacent part to the end of the villus in the whole villus, including the villus base part.

[0050] Furthermore, the present invention provides a tissue regeneration method, which includes the step of treating an individual in need thereof using chorionic plate adjacent villus tissue-derived stem cells derived from chorionic plate adjacent villus tissue, wherein the chorionic plate adjacent villus tissue is located in the area from the chorionic plate adjacent part to 1 / 3 of the distance from the chorionic plate adjacent part to the end of the villus in the whole villus, including the villus base part.

[0051] Furthermore, the present invention provides a tissue regeneration method, which comprises the following steps: 1) obtaining villi adjacent to the chorionic plate by cutting the chorionic plate adjacent portion from the whole villus to an area from the chorionic plate adjacent portion to 1 / 3 of the length of the whole villus; 2) obtaining stem cells from the villi adjacent to the chorionic plate; and 3) using the obtained chorionic plate adjacent villus-derived stem cells to treat an individual in need thereof.

[0052] In the present invention, the above-mentioned treatment includes all actions such as administration and application, and preferably, may mean an action of directly injecting into a site in need of tissue regeneration of an individual in need of treatment.

[0053] The chorionic plate adjacent villus tissue-derived stem cells of the present invention not only exhibit excellent colony-forming ability and excellent proliferation ability, but can also be differentiated into a variety of cell types such as adipocytes, chondrocytes, bone cells, nerve cells, ligament cells or tendon cells (tenocytes), thereby being able to be used as cell therapy agents for tissue regeneration and compositions for tissue regeneration.

[0054] In the present invention, "cellular therapeutic agent" refers to a drug that is isolated, cultured and specially processed from the human body and is used for the treatment, diagnosis and prevention of cells and tissues (as stipulated by the U.S. FDA). It refers to a drug that uses such cells for the treatment, diagnosis and prevention of diseases through a series of actions such as in vitro proliferation and screening of living autologous, allogeneic or xenogeneic cells or changing the biological characteristics of cells by other methods in order to restore the function of cells or tissues.

[0055] The chorionic plate adjacent villus tissue-derived stem cells of the present invention can be used for target cell populations of body tissues or organs, for example, can be used for various treatment schemes for adjustment, enhancement, treatment or replacement by implantation, transplantation or injection of stem cells or differentiated cell populations. The chorionic plate adjacent villus tissue-derived stem cells of the present invention can make the tissue become a new tissue or a changed tissue by replacing or enhancing the existing tissue, or can make it combined with biological tissues or structures.

[0056] Preferably, the cell therapy agent of the present invention can be used to regenerate one or more tissues selected from the group consisting of fat, cartilage, bone, nerve, ligament and tendon. In addition, in the present invention, cartilage may include hyaline cartilage, fibrocartilage or elastic cartilage without limitation, and the above cartilage may be selected from the group consisting of articular cartilage, ear cartilage, nasal cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage and spinal cartilage.

[0057] Therefore, the cell therapeutic agent of the present invention can be used to treat bone defects, tendon and ligament defects, fat tissue defects, cartilage necrosis, osteochondritis, cartilage rupture, cartilage damage, arthritis, cartilage defects or congenital tracheomalacia.

[0058] Furthermore, the cell therapy agent or composition for tissue regeneration of the present invention can be directly administered into a joint or joint cavity, in which case, it can be administered in the form of an injection. Since the chorionic plate adjacent villus tissue-derived stem cells of the present invention are stem cells with excellent differentiation ability that can differentiate into cartilage, bone, fat, etc., various lesions, such as lesions of articular cartilage, can be treated or prevented by administering them to a joint site, cartilage site, tendon site, or ligament site. In particular, if the chorionic plate adjacent villus tissue-derived stem cells of the present invention are administered into a joint, cartilage site, tendon site, or ligament site, cartilage can be regenerated in a state where the cartilage surface is smooth and almost undamaged, and the cartilage can be protected and regenerated to prevent the progression of arthritis.

[0059] In particular, the cell therapeutic agent or composition for tissue regeneration of the present invention can be formulated as an injectable formulation for direct administration to the chorionic plate-adjacent villus tissue-derived stem cells, in which case, a hydrogel may be further included for suitability for injection.

[0060] The hydrogel that can be used in the cell therapy agent of the present invention may include, without limitation, hydrogels known in the art that are suitable for injection, and may be one or more selected from the group consisting of small intestinal submucosa, hyaluronic acid, carboxymethylcellulose (CMC), alginate, chitosan, polyacrylamide, poly(N-isopropylacrylamide), β-sodium glycerophosphate (β-Pluronic, Poly(ethylene oxide)poly(propylene oxide)poly(ethyleneoxide)), fibrin, polyethylene oxide (PEO), and a mixture of carboxymethylcellulose and polyethyleneimine (PEI). In a preferred embodiment of the present invention, hyaluronic acid and Pluronic can be mixed with villous tissue-derived stem cells adjacent to the chorionic plate for use.

[0061] The preferred dosage of the cell therapy agent of the present invention varies according to the individual's condition, body weight, severity of the disease, drug form, route of administration and time, and can be appropriately selected by a person skilled in the art. Administration can be once a day or in divided doses, and the above dosage does not limit the scope of the present invention at all levels.

[0062] Furthermore, the present invention provides the use of stem cells derived from villus tissue adjacent to the chorionic plate in the preparation of tissue regeneration therapeutic agents, characterized in that the villi adjacent to the chorionic plate are the 1 / 3 region from the chorionic plate adjacent portion in the whole villus to the distance from the chorionic plate adjacent portion to the end of the villus, including the villus base portion.

[0063] Furthermore, the present invention provides the use of stem cells derived from chorionic plate adjacent villus tissue in tissue regeneration, characterized in that the chorionic plate adjacent villus is a region from the chorionic plate adjacent part in the whole villus to 1 / 3 of the distance from the chorionic plate adjacent part to the end of the villus, including the villus base part.

[0064] The above-mentioned chorionic plate adjacent villus tissue-derived stem cells are stem cells with excellent differentiation ability, which can differentiate into cartilage, bone, fat, etc., and can be administered to joints, cartilage, tendons or ligaments to treat or prevent various lesions, for example, lesions of articular cartilage. In particular, if the chorionic plate adjacent villus tissue-derived stem cells of the present invention are administered into a joint, the cartilage surface can be regenerated in a state where the cartilage surface is smooth and almost undamaged, and the cartilage can be protected and regenerated to prevent the progression of arthritis. Therefore, the above-mentioned tissue regeneration therapeutic agent can be used to treat bone defects, tendon and ligament defects, adipose tissue defects, cartilage necrosis, osteochondritis, cartilage rupture, cartilage damage, arthritis, cartilage defects or congenital tracheomalacia, and the above-mentioned tissue regeneration use can be cartilage regeneration.

[0065] Furthermore, the present invention provides a cell therapy agent or a composition for tissue regeneration, wherein the cell therapy agent for tissue regeneration comprises stem cells derived from villi adjacent to the chorionic plate, which are obtained by a method for isolating or preparing stem cells derived from villi adjacent to the chorionic plate, the method comprising: 1) cutting the chorionic plate adjacent portion of the whole villus to an area from the chorionic plate adjacent portion to 1 / 3 of the length of the whole villus to obtain villi adjacent to the chorionic plate; and 2) obtaining stem cells from villi adjacent to the chorionic plate; the composition for tissue regeneration comprises stem cells derived from villi adjacent to the chorionic plate, which are obtained by a method for isolating or preparing stem cells derived from villi adjacent to the chorionic plate, the method comprising: 1) cutting the chorionic plate adjacent portion of the whole villus to an area from the chorionic plate adjacent portion to 1 / 3 of the length of the whole villus to obtain villi adjacent to the chorionic plate; and 2) obtaining stem cells from villi adjacent to the chorionic plate.

[0066] Furthermore, the present invention provides a tissue regeneration method, which comprises the following steps: 1) cutting the area from the chorionic plate adjacent part to the 1 / 3 position in the length of the whole villus to obtain the villi adjacent to the chorionic plate; 2) obtaining stem cells from the villi adjacent to the chorionic plate; and 3) using the obtained villus-derived stem cells adjacent to the chorionic plate to treat an individual in need thereof.

[0067] The chorionic plate adjacent villus tissue-derived stem cells of the present invention have not only excellent proliferation and differentiation abilities, but also excellent tissue regeneration effects. In order to avoid complicating the specification, the contents that overlap with those described in the cell therapy agent for tissue regeneration will be omitted below.

[0068] Hereinafter, the present invention will be further described in detail by the following examples. Such examples are only used to illustrate the present invention in detail, and the scope of the present invention is not limited to such examples.

[0069] Embodiments of the present invention

[0070] Example 1: Isolation of adjacent villi of the placental chorionic plate and acquisition of stem cells

[0071] After removing the amnion from the placental tissue and removing the decidua with sterile scissors, the whole trophoblast (chorion) of about 5cm×5cm×3cm in size was obtained and transferred to a 150mm culture dish, and then washed with phosphate buffered saline (PBS) for more than 5 times to remove blood and blood cells. In order to separate only the villi, the whole trophoblast was separated into villi and intervillous again, and the separated villi were washed more than 5 times with phosphate buffered saline to remove the remaining blood and blood cells. Only the chorionic tissue corresponding to the chorionic plate adjacent part (the chorionic plate adjacent part in the whole villi to 1 / 5 of the distance from the chorionic plate adjacent part to the end of the villi) was cut and obtained from the washed whole villi using sterile forceps and a scalpel. In order to distinguish it from the whole villi (CV, chorionic villi), the cut part of the chorionic tissue including the base of the villi obtained in this way was defined as the chorionic plate adjacent villi. The adjacent villi of the chorionic plate Figure 1 shown.

[0072] After transferring the adjacent villi of the chorionic plate (hereinafter referred to as VCP) to a 50 ml test tube, a-MEM culture medium supplemented with 0.2% collagenase was added and reacted at 37°C for 2 hours using a stirrer to obtain cells derived from VCP. The obtained VCP-derived cells were filtered using a 100 μm filter to remove undecomposed tissue, and after adding a-MEM culture medium supplemented with fetal bovine serum and antibiotics, centrifuged at 1500 rpm for 4 minutes at a temperature of 25°C. After removing the supernatant, a-MEM culture medium without growth factors and supplemented with fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at a temperature of 37°C and 5% carbon dioxide. Subsequently, cells attached to the bottom of the culture container were screened from the above culture to obtain VCP-derived stem cells. The obtained stem cells are called "CV1".

[0073] Comparative Example 1: Acquisition of stem cells derived from other tissues

[0074] 1-1. Obtaining stem cells derived from whole placenta

[0075] The whole placental tissue was finely cut into about 5cm×5cm×3cm and the placental tissue was washed with phosphate buffer to remove blood and blood cells. A-MEM culture medium supplemented with 0.2% collagenase was added to the washed placental tissue and reacted at 37°C for 2 hours using a stirrer to obtain placental cells. The obtained placental cells were filtered using a 100μm filter to remove undecomposed tissue, and after adding a-MEM culture medium supplemented with fetal bovine serum and antibiotics, centrifuged at 1500rpm for 4 minutes at a temperature of 25°C. After removing the supernatant, a-MEM culture medium without growth factors and supplemented with fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at a temperature of 37°C and 5% carbon dioxide. Subsequently, cells attached to the bottom of the culture container were screened from the above culture to obtain whole placenta (Pla, Whole placenta) derived stem cells.

[0076] 1-2. Obtaining stem cells derived from local placental tissue

[0077] After removing the amnion and decidua from the placental tissue, a trophoblast of approximately 5 cm × 5 cm × 3 cm in size was obtained and transferred to a 150 mm culture dish, and then washed more than 5 times with phosphate buffer to remove blood and blood cells. In order to separate only the villi, the whole trophoblast was separated into villi and intervillous again, and the separated villi were washed more than 5 times with phosphate buffer to remove the remaining blood and blood cells. The remaining tissue portion of the whole villi (CV, chorionic villi) except for the chorionic plate adjacent villi recorded in Example 1 is defined as CV-VCP as the meaning of excluding the chorionic plate adjacent villi from the whole villi. After transferring the whole villi or CV-VCP tissue to a 50 ml test tube, a-MEM culture medium supplemented with 0.2% collagenase was added and reacted at 37°C for 2 hours using a stirrer to obtain cells derived from each tissue. The obtained cells were filtered using a 100 μm filter to remove undecomposed tissues, and after adding a-MEM medium supplemented with fetal bovine serum and antibiotics, centrifuged at 1500 rpm for 4 minutes at a temperature of 25°C. After removing the supernatant, a-MEM medium without growth factors and supplemented with fetal bovine serum and antibiotics was added to the remaining precipitated cells, and cultured at a temperature of 37°C and 5% carbon dioxide. Subsequently, cells attached to the bottom of the culture container were screened from the above culture, and stem cells derived from the whole villi of the trophoblast were called "CV", and stem cells derived from CV-VCP were called "CV2".

[0078] Example 2: Culture and cell morphology of chorionic plate adjacent villus-derived stem cells CV1

[0079] After washing the CV1 stem cells obtained by the above-mentioned Example 1 with phosphate buffer, replace the a-MEM culture medium without growth factors and supplemented with fetal bovine serum and antibiotics every 2 to 3 days for culture. At the time point when the above-mentioned stem cells grow by more than 80%, the stem cells are separated from the culture container by treatment with a pancreatic enzyme substitute (TryPLE), and the separated stem cells are diluted at a ratio of 1 / 5, and then subcultured by culturing in other culture containers. Subsequently, the cell morphology after culture is observed under a microscope. In addition, the whole placenta and other local tissue-derived stem cells obtained by the above-mentioned Comparative Example 1 are subcultured by the same method, and then the cell morphology after culture is observed under a microscope. The results are as follows. Figure 2 shown.

[0080] like Figure 2 As shown, it can be confirmed that the CV1 stem cells of the present invention exhibit morphological characteristics of fibroblasts and specifically maintain only single cells. In contrast, although whole placenta and other local tissue-derived stem cells exhibit morphological characteristics of fibroblasts, they are composed of a mixture of multiple cells with different morphologies.

[0081] That is, compared with the CV1 stem cells of the present invention, although the CV1 stem cells specifically maintain only a single cell before and after subculture, the stem cells derived from the whole placenta and other local tissue-derived stem cells are a mixture of cells of different morphologies.

[0082] Example 3: Analysis of colony-forming ability of CV1 stem cells derived from adjacent villi of the chorionic plate

[0083] In the above Example 1, the colony-forming ability of CV1 stem cells was confirmed. More specifically, the CV1 stem cells obtained from the above Example 1 were subjected to the first subculture by the method of the above Example 2. At the end of the above subculture, 5×10 3 After that, they were cultured in a-MEM medium without growth factors and supplemented with fetal bovine serum and antibiotics for 14 days. Subsequently, the number of colonies formed by each stem cell was counted. In addition, the population doubling time and colony formation ability of the whole placenta and other placental local tissue-derived stem cells obtained in the above-mentioned comparative example 1 were measured by the same method. In the process of measuring the colony formation ability, the result value of the whole placenta-derived stem cells was converted to 100%. The results are shown in Figure 2. Figure 3 and Figure 4 shown.

[0084] like Figure 3As shown in Part A of the Figure 1, it can be confirmed that CV1 stem cells derived from VCP have significantly better colony-forming ability than whole placenta (Pla), whole villus-derived (CV), and CV-VCP-derived CV2 stem cells in whole villi excluding the villi adjacent to the chorionic plate. Figure 3 As shown in Part B, it was confirmed that the colony size formed by CV1 stem cells derived from VCP was 3 mm to 4 mm or more.

[0085] Therefore, compared with CV2 stem cells, CV1 exhibits more than twice the colony-forming ability, and compared with whole villus and other villus-derived stem cells, CV1 is a stem cell with excellent colony-forming ability.

[0086] And, if Figure 4 As shown, the population doubling time was similar from P2 to P4, but the CV1 stem cells of the present invention showed the lowest population doubling time after P5. Based on this result, it was confirmed that the stem cells CV1 derived from the villi adjacent to the chorionic plate are stem cells with excellent proliferation ability.

[0087] Example 4: Analysis of surface markers of chorionic plate adjacent villus-derived stem cells CV1

[0088] In order to confirm the immune characteristics of CV1 obtained in the above Example 1, surface marker analysis was performed using a 242-panel human cell surface marker screening panel.

[0089] First, the adjacent villi of the chorionic plate were obtained by Example 1 and washed with phosphate buffer. Subsequently, the stem cells were recovered after treatment with pancreatic enzyme substitute and centrifuged at 1200 rpm for 5 minutes. After removing the supernatant, the stem cells were washed with phosphate buffer and then centrifuged at 1200 rpm for 5 minutes. After removing the supernatant, the stem cells were suspended in phosphate buffer and passed through a 40 μm cell strainer, and then the cells and BD Pharmingen Stain Buffer + EDTA were prepared for mixing. 100 μl (100,000 cells / well) was inoculated into each well of a round-bottom 96-well plate (Cat. 353910). Next, 20 μl of the corresponding antibody was inoculated into each well. 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. After carefully removing the supernatant again, wash with BD Pharmingen Stain Buffer + EDTA and centrifuge at 1200 rpm for 5 minutes. After that, 100 μl of secondary antibody was inoculated into each well. After adding BD Pharmingen Stain Buffer + EDTA to each well for washing, centrifuge at 1200 rpm for 5 minutes, remove the supernatant and repeat the washing process twice carefully using phosphate buffer. Remove the supernatant and dispense 150 μl of BD Pharmingen Stain Buffer + EDTA into each well. Surface markers of more than 10,000 cells per well were analyzed using flow cytometry (FACS). In addition, surface markers of whole placenta and other placental local tissue-derived stem cells obtained in Comparative Example 1 were analyzed by the same method. The results are shown in Tables 1 and 2.

[0090] Table 1

[0091] Unit: %

[0092]

[0093] Table 2

[0094] unit:%

[0095]

[0096] When the surface marker expression is less than 5%, it is classified as a negative marker, and when the surface marker expression is 6% or more, it is classified as a positive marker. Positive markers are classified into low (6% or more and less than 30%), middle (30% or more and less than 70%), and high (70% or more) based on their expression. The marker expression values ​​in Tables 1 and 2 are sorted according to this standard and the marker expression is shown in Tables 3 and 4.

[0097] Table 3

[0098]

[0099] Table 4

[0100]

[0101] As shown in Table 3 above, it was confirmed that CV1 of the present invention was negative for the markers CD273, CD275, and CD321, which were different from the whole placenta and chorionic villus-derived stem cells CV. In particular, this expression was also different from the results of CD2, a chorionic villus CV-VCP-derived stem cell other than the villi adjacent to the chorionic plate. Therefore, it was confirmed that it was a new type of stem cell that showed completely different CD marker expression characteristics compared to other stem cells.

[0102] Furthermore, the above results indicate that stem cells derived from whole villi are a mixture of stem cells showing various CD marker expression patterns. Therefore, it was confirmed that more uniform stem cells can be obtained by subdividing and isolating stem cells from adjacent villi of the chorionic plate as in the present invention.

[0103] Example 5: Comparison of the cell sources of CV1 stem cells derived from adjacent villi of the chorionic plate

[0104] An experiment to confirm whether or not CV1 obtained from Example 1 above belongs to other cell populations of different cell origin was performed by comparing CV1 with whole placenta or chorionic villus-derived stem cells and CV2 with chorionic villus CV-VCP derived stem cells excluding chorionic plate adjacent villi.

[0105] The placenta is a special organ in which cells derived from the mother and the fetus are mixed. There are cells derived from the mother and the fetus in the placenta. Generally, it is known that the trophoblast is derived from the mother and the villi are derived from the fetus. Thus, an experiment for confirming the cell source of the cells of CV1 obtained from Example 1 and Comparative Example 1 was performed, and the cells obtained from P5, which is widely used in clinical experiments, were smeared on a slide and the X chromosome and Y chromosome were stained with a probe using markers Cy3 (red) and fluoresceinisothiocyanate (green) (Vysis, Downers Grove, IL, USA). After the slide analysis was stained with DAPI for cell nuclei, the spectral red and green filters at X1000 magnification were used for confirmation, and then the cell source was confirmed by counting the X chromosomes and Y chromosomes expressed by the cells, and the results are shown in Table 5.

[0106] Table 5

[0107]

[0108] As shown in Table 5, in the case of Pla, although XX and XY were mixed in the initial passage, 100% XX or 100% XY was confirmed at P5, and XY was confirmed in the cases of CV and CV1. In contrast, CV2 was derived from 100% XX cells at P5.

[0109] That is, as shown in Table 5, CV1 cells show a chromosome type of cells derived from the fetus, not a chromosome type of cells derived from the mother, whereas CV2 cells show a chromosome type derived from the mother, and are stem cells having different cell origins.

[0110] Example 6: Confirmation of the Chondrocyte Differentiation Ability of Chorionic Plate Adjacent Villi Derived Stem Cells CV1

[0111] In order to confirm the differentiation ability of the chorionic plate adjacent villus-derived stem cells CV1 obtained from the above-mentioned Example 1 into chondrocytes, the stem cells were cultured in a known chondrocyte induction differentiation medium (DMEM medium containing 0.1 μM dexamethasone, 50 μg / ml ascorbic acid, 40 μg / ml L-proline, 10 ng / ml transforming growth factor β3, 500 ng / ml bone morphogenetic protein 6, and 50 mg / ml ITS premix) for 3 weeks to induce differentiation into chondrocytes. In order to determine the degree of differentiation of the above-mentioned stem cells into chondrocytes, the immunochemical staining method using Safranin-O staining and type II collagen was performed by existing known methods. In addition, after 3 weeks of culture, the sulfated glycosaminoglycan detection kit (Biocolor Ltd., Carrickfergus, County Antrim, UK) was used to determine the content of sulfated glycosaminoglycan (GAG). The absorbance of the 96-well plate at a wavelength of 656 nm was measured using an enzyme reader, and the results are as follows. Figure 5 shown.

[0112] like Figure 5 As shown, it was confirmed that the CV1 stem cells of the present invention have a superior chondrocyte differentiation ability compared to CV and CV2.

[0113] Example 7: Confirmation of osteoblast differentiation ability of chorionic plate adjacent villus-derived stem cells CV1

[0114] In order to confirm the differentiation ability of the chorionic plate adjacent villus derived stem cells CV1 obtained from the above Example 1 into osteocytes, the stem cells were cultured in a known osteocyte induction differentiation medium (DMEM medium containing 10% fetal bovine serum, 1% antibiotics, 100 μM dexamethasone, 50 mM ascorbic acid-2-phosphate trisodium salt, 10 μM sodium β-glycerophosphate, and 250 μM ascorbic acid) for 4 weeks to induce differentiation into osteocytes. In this case, after the start of differentiation induction, at the time point of 2 weeks, the alkaline phosphatase (ALP) staining method of the known method was used for staining, and at the time point of 4 weeks, the alizarin red S staining method of the known method was used for staining to analyze the degree of differentiation into osteocytes. Furthermore, the differentiation ability of the whole placenta and other placental local tissue-derived stem cells obtained from the above-mentioned Comparative Example 1 into osteocytes was determined by the same method. After 4 weeks, the cells were treated with 0.6M hydrogen chloride for 24 hours and stored at 37°C to measure intracellular calcium. The absorbance was measured at 565nm using the o-cresolphthalein complexone method (Pointe Scientific, Canton, MI, USA), and then the calcium concentration was standardized and quantified. The results are shown in FIG. Figure 6 shown.

[0115] like Figure 6 As shown, it was confirmed that the chorionic plate adjacent villus-derived stem cells CV1 of the present invention have a more excellent osteocyte differentiation ability to differentiate into osteocytes than CV and CV2.

[0116] Example 8: Confirmation of the ability of chorionic plate adjacent villus derived stem cells CV1 to differentiate into adipocytes

[0117] In order to confirm the differentiation ability of the chorionic plate adjacent villus-derived stem cells CV1 obtained from the above-mentioned Example 1 into adipocytes, the hepatocytes were cultured for 3 weeks by replacing the known adipocyte induction differentiation medium 1 (DMEM medium containing 10% fetal bovine serum, 1% antibiotics, 1μM dexamethasone, 20μM indomethacin, 10μM insulin, and 50μM 3-isobutyl-1-methylxanthine) and adipocyte induction differentiation medium 2 (DMEM medium containing 10% fetal bovine serum, 1% antibiotics, and 10μM insulin) every 3 to 4 days to induce differentiation into adipocytes. In order to determine the degree of differentiation of the above-mentioned stem cells into adipocytes, Oil red O staining was performed by the existing known method. In addition, the ability of the whole placenta, other placental local tissue-derived and other tissue-derived stem cells obtained from Comparative Example 1 to differentiate into adipocytes was determined by the same method. The lipids were dissolved by treatment with 100% isopropanol solution and the absorbance at 500nm was measured. The results are as follows. Figure 7 shown.

[0118] like Figure 7 As shown, it was confirmed that CV1 of the present invention has a superior adipocyte differentiation ability compared to CV and CV2.

[0119] Example 9: Chorionic plate adjacent villus derived stem cells CV1 as cell therapy in an animal model of arthritis Verification of the effect

[0120] In order to verify the effect of the chorionic plate adjacent villus derived stem cell CV1 obtained from the above-mentioned Example 1 as a cell therapy agent in the cartilage injury animal model, the following experiment was performed. More specifically, in order to prepare an arthritis animal model using Wistar rats (rats, male, 12 weeks old), healthy rats were selected and injected with an appropriate amount of ketamine and dimethylbenzyl hydrochloride for anesthesia according to their body weight. After confirming that the rats were fully anesthetized, the knee joints of the lower limbs on both sides were shaved and fixed with a bandage while maintaining the posture. After disinfecting the knee joints on both sides with povidone and determining the position of the patella by palpation, the incision lines along the upper side, lower side, and medial side of the knee joint were passed through the paramedian approach to the medial side of the knee joint, and then the inside of the joint was observed by moving the patella outward and bending the knee joint. After confirming that there were no specific pathological results, a medial meniscus injury model (DMM, destabilization of the medial meniscus) was prepared by damaging the meniscus. Eight weeks after the injury was induced in the above manner, 1 ml of CV1 stem cell suspension was mixed with 5% hyaluronic acid and 10% Pluronic using a syringe, and 20 μl was injected into the joint cavity of the above animal model. After confirming that the rat had awakened from anesthesia, the rat was allowed to move freely. In order to prevent infection after the operation, analgesics and antibiotics were administered for 3 consecutive days. After 8 and 12 weeks of administration, sections were obtained from the injured and treated joints of the rats, respectively, and H&E and Safranin O staining were performed. The degree of regeneration of the newly formed cartilage was quantified by using the OARSI score. The results are as follows. Figure 8 and Fig. 9 shown.

[0121] like Figure 8 As shown, compared with the control group injected with saline after 8 and 12 weeks, it was confirmed that the injected CV1 stem cells regenerated the cartilage in a state where the surface of the cartilage became smooth and there was no cartilage damage.

[0122] And, if Fig. 9 As shown in the OARSI score results, after 8 weeks, the score of the CV1 stem cell injection group (3.8±0.8) was lower than that of the control group (8.6±0.5), and even after 16 weeks, the score of the CV1 stem cell injection group (3.0±0.7) was lower than that of the control group (9.6±0.5). Therefore, CV1 stem cells have the effect of protecting and regenerating cartilage during the progression of arthritis to prevent further progression of arthritis.

[0123] The above experimental results confirm that, since the existing stem cells derived from the whole placenta are a mixture of stem cells derived from local tissues with various characteristics and cannot uniformly show the ability to differentiate into different cells, the stem cells CV1 derived from the chorionic plate adjacent villi, which is a local tissue of the placenta, of the present invention have better characteristics in terms of excellent differentiation ability and homogeneity of various characteristics of other stem cells than the existing whole placenta-derived stem cells. In particular, in terms of growth, proliferation, morphology and differentiation characteristics, the CV1 stem cells of the present invention show a consistent morphology and the best stem cell characteristics compared to other placental local tissue-derived stem cells. Therefore, the above-mentioned CV1 stem cells can not only improve the differentiation efficiency to target cells, but also can be effectively used as a cell therapy for various diseases.

Claims

1. A stem cell derived from the villous tissue adjacent to the chorionic plate, characterized in that: The chorionic plate adjacent villous tissue is a continuous part in the whole villus starting from the chorionic plate adjacent part to 20% of the whole villus length, and includes the villus base part, wherein the stem cells have negative surface factor expression characteristics for CD273, CD275 or CD321, and The stem cells have the following surface marker expression characteristics: i) positive for CD44, CD73, CD90 and CD105; ii) positive for CD49e, CD140b or CD142; and iii) Negative for CD31, CD34, CD45 and HLA-DR.

2. The stem cells derived from the villous tissue adjacent to the chorionic plate according to claim 1, characterized in that: The stem cells are stem cells derived from embryonic cells.

3. The stem cells derived from the villous tissue adjacent to the chorionic plate according to claim 1, characterized in that: The stem cells can be obtained by the following steps: 1) Cut the area from the chorionic plate adjacent part to 1 / 5 of the distance from the chorionic plate adjacent part to the end of the chorionic villus to obtain the chorionic plate adjacent villus; as well as 2) Obtain stem cells from the villi adjacent to the chorionic plate.

4. The method for isolating and obtaining stem cells derived from the villous tissue adjacent to the chorionic plate according to claim 1, characterized in that: The steps include: 1) obtaining chorionic villus tissue adjacent to the chorionic plate in the whole villus, from the chorionic plate adjacent site to a region 1 / 5 of the distance from the chorionic plate adjacent site to the villus end, The chorionic plate adjacent villus tissue is a continuous part of the whole villus starting from the chorionic plate adjacent part to 20% of the whole villus length; and 2) obtaining stem cells from the villus tissue adjacent to the chorionic plate, wherein the stem cells obtained from the villus tissue adjacent to the chorionic plate have negative surface factor expression characteristics for CD273, CD275 or CD321.

5. A cell therapy agent for cartilage regeneration, characterized in that: The composition comprises the stem cells according to claim 1 as an effective ingredient.

6. The cell therapy agent according to claim 5, characterized in that The cartilage is hyaline cartilage, fibrous cartilage or elastic cartilage.

7. The cell therapy agent according to claim 5, characterized in that The cartilage is selected from ear cartilage, nasal cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage and spinal cartilage.

8. The cell therapy agent according to claim 5, characterized in that The cell therapy agent is an injection.

9. The cell therapy agent according to claim 5, characterized in that The cell therapy agent further comprises a hydrogel.

10. The cell therapy agent according to claim 9, characterized in that The hydrogel is one or more selected from a mixture of small intestinal submucosal tissue, hyaluronic acid, carboxymethyl cellulose, alginate, chitosan, polyacrylamide, poly-N-isopropylacrylamide, sodium β-glycerophosphate, pluronic, fibrin, polyethylene oxide, carboxymethyl cellulose and polyethyleneimine.

11. Use of the stem cells according to claim 1 as an effective ingredient in preparing a composition for cartilage regeneration.

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