KR20220130627A - Stem cells derived from intervillous space adjacent to decidua and cellular therapeutic agents comprising the same - Google Patents

Abstract

The present invention relates to stem cells derived from a tissue from the intervillous space, preferably the intervillous space adjacent to decidua, and to a cellular therapeutic agent comprising the same. The stem cells derived from the intervillous space adjacent to decidua exhibits homogeneous growth characteristics and excellent proliferation characteristics compared to other placental tissue-derived stem cells, and has a remarkably excellent differentiation ability into cartilages, bones, and fat, and thus can be effectively used for various tissue regeneration treatments that require regeneration of cartilages, bones and fat, especially cartilage regeneration and osteoarthritis treatment.

Description

Stem cells derived from intervillous space adjacent to decidua and cellular therapeutic agents comprising the same}

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. 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.

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.

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.

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.

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.

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.

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.

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.

In addition, 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.

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.

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.

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. .

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.

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).
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.
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).
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).
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).
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.

Hereinafter, the present invention will be described in detail.

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. .

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.

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.

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'.

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.

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.

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.

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

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.

In 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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

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.

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.

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.

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.

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.

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.

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. 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.

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.

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.

Example 1: Separation of the interchorionic cavity adjacent to the placental decidua and obtaining stem cells

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 .

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. Cells attached to the bottom of the culture vessel were selected from the culture to obtain stem cells derived from CT-V2.

Comparative Example 1: Obtaining stem cells derived from other tissues

1-1. Obtaining stem cells derived from whole placenta

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. Obtaining Stem Cells Derived from Placental Details

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.

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.

Example 2: Decidual adjacent interchorionic space (CT-V2) derived stem cell culture and cell morphology confirmation

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.

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 .

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.

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.

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 CT-V2-derived stem cells obtained in Example 1 were subjected to the first subculture 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 formation performance were measured in the same manner. In the case of colony-forming ability, the result value of whole placental-derived stem cells was converted to 100%. The results are shown in FIGS. 3 and 4 .

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.

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.

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.

Example 4: Analysis of surface markers of stem cells derived from decidual adjacent interchorionic space (CT-V2)

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.

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.

[Table 1]

[Table 2]

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.

[Table 3]

[Table 4]

As shown in Table 3, CT-V2 of the present invention was confirmed to have 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 were negative. It was confirmed that the 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.

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.

Example 5: Confirmation of differentiation ability into adipocytes of stem cells CT-V2 derived from the interchorionic space adjacent to the decidua

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 .

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.

Example 6: Confirmation of chondrocyte differentiation ability of stem cells derived from decidua adjacent interchorionic space (CT-V2)

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 .

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.

Example 7: Confirmation of differentiation ability of stem cells derived from decidua adjacent interchorionic space (CV-V2) into osteocytes

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 .

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.

Example 9: Verification of cell therapeutic effect of stem cells derived from the decidua adjacent chorionic space (CT-V2) (cartilage defect animal model)

In order to verify the effect of stem cells derived from the decidua 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-damaged 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 the top, bottom, and medial side of the patella to reach the inside of the knee joint in 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 was not naturally healed.

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.

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

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.

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.

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.

As a result of type II collagen staining, it was confirmed that it was evenly distributed in the tissue regeneration site.

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

Therefore, it was found that CT-V2 stem cells appear to play a role in regenerating damaged cartilage.

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 (18)

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.
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.
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.
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.
According to claim 1, wherein the stem cells are stem cells of parental cell origin, stem cells.
The method of claim 1, wherein the stem cells are
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; Characterized in that obtained through, stem cells.
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; A method for separating and obtaining stem cells derived from the interchorionic cavity adjacent to the decidua comprising a.
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.
The method of claim 7, wherein the stem cells have positive surface factor expression characteristics for CD49b, CD74 or CD200.
A cell therapy product for tissue regeneration comprising the stem cell of claim 1 as an active ingredient.
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.
The cell therapeutic agent for tissue regeneration according to claim 11, wherein the cartilage is free cartilage, fibrocartilage or elastic cartilage.
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.
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.
The cell therapy for tissue regeneration according to claim 10, wherein the cell therapy for tissue regeneration is an injection.
The cell therapy for tissue regeneration according to claim 15, wherein the cell therapy for tissue regeneration further comprises a hydrogel.
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).
A composition for tissue regeneration comprising the stem cell of claim 1 as an active ingredient.

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