Trophoblast sublineage-derived stem cells and tissue regeneration composition comprising same

Abstract

The present invention relates to trophoblast sublineage-derived stem cells and a tissue regeneration composition comprising same. Compared to stem cells derived from other trophoblast layers, the stem cells exhibiting a novel CD marker expression pattern, according to the present invention, have superior cell proliferation ability and remarkably superior differentiation ability into various tissues, and thus can be effectively used in tissue regeneration treatment, particularly, in cartilage regeneration and osteoarthritis treatment.

Description

Stem cells derived from trophoblast tissue and a composition for tissue regeneration containing the same

[0001] The present invention relates to stem cells derived from trophoblast tissue and a composition for tissue regeneration containing the same.

[0002] The placenta is a disc-shaped organ that develops in the uterine wall during pregnancy. Rich in blood vessels, it is the foetus's primary source of nutrition, respiration, and excretion. With scientific research demonstrating that lifelong health is determined during the fetal period, the importance of the placenta during pregnancy is being highlighted, and significant efforts are being made to elucidate the interrelationships between various substances. The placenta is composed of diverse cell types, depending on the week and location of the pregnancy, yet its application remains extremely limited.

[0003] Recently, research is also underway on stem cells isolated from placental tissue. Placental stem cells can yield 100 times more mesenchymal stem cells than umbilical cord blood, and they are known to be reusable, even into adulthood. Furthermore, unlike cord blood hematopoietic stem cells, which are primarily used for blood diseases, placental stem cells can be used as therapeutics for a variety of diseases that involve cell damage or require regeneration.

[0004] Previously, most placental tissue-derived stem cells were derived from a portion of the full-term placenta that could be obtained at birth. However, the placenta is composed of various tissues, such as the basement membrane attached to the uterus, the villous tissue that is the central tissue of the placenta and mediates the exchange of substances between the maternal and fetal blood, and the chorion that covers the inside of the placenta and envelops the fetus and amniotic membrane. However, research on these detailed tissues has not yet been widely known. Meanwhile, Korean Patent Registration No. 818214 discloses a method for isolating stem cells from the amnion or decidua using a medium containing NAC (N-acrtyl-L-cysteine), and Korean Patent Registration No. 871984 discloses the multipotency of stem cells derived from amnion, serosa, basal decidua, and placental tissue using a medium containing bFGF (Basic Fibroblast Growth Factor).

[0005] However, the stem cells obtained from the segmented part of the placenta and their effects have not been widely reported yet, regardless of the existing anatomically known method of distinguishing the placental structure.

[0006] Accordingly, the inventors of the present invention completed the present invention by identifying stem cells derived from trophoblast tissues that have high expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 and low expression of CD10, CD107b, or CD340 markers compared to whole trophoblast-derived stem cells while studying stem cells that can be used for tissue regeneration.

[0007] Accordingly, the purpose of the present invention is to provide the novel stem cells, a tissue regeneration composition containing the same, a cell therapy agent, and a method for producing the same.

[0008]

[0009] To achieve the above purpose, the present invention provides stem cells derived from trophoblast tissues, which have higher expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b and CD273 and lower expression of CD10, CD107b or CD340 markers compared to whole trophoblast-derived stem cells.

[0010] Additionally, the present invention provides a composition for tissue regeneration containing the stem cells as an active ingredient.

[0011] In addition, the present invention provides a cell therapeutic agent for tissue regeneration containing the above stem cells as an active ingredient.

[0012] In addition, the present invention includes a method for obtaining stem cells, including the steps of: 1) dividing the entire trophoblast layer into 10 parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and obtaining tissue corresponding to the 1 / 10 part closest to the chorionic plate; and 2) obtaining stem cells from the obtained tissue; wherein the stem cells provide a method for obtaining stem cells derived from trophoblast detailed tissues, wherein the stem cells have high expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 and low expression of CD10, CD107b, or CD340 markers compared to the entire trophoblast-derived stem cells.

[0013] In addition, the present invention provides a method for producing a composition for tissue regeneration, comprising: 1) dividing the entire trophoblast layer into 10 parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and obtaining tissue corresponding to the 1 / 10 part closest to the chorionic plate; 2) obtaining stem cells from the obtained tissue; and 3) mixing the obtained stem cells with a hydrogel;

[0014] The present invention provides a method for producing a composition for tissue regeneration, wherein the stem cells are stem cells having high expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b and CD273 and low expression of CD10, CD107b or CD340 markers compared to whole mesenchymal stem cells.

[0015] In addition, the present invention provides a tissue regeneration method comprising a step of administering stem cells derived from trophoblast sub-tissue to an individual in need thereof, wherein the stem cells derived from trophoblast sub-tissue are characterized in that, compared to whole trophoblast-derived stem cells, the expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 is high, and the expression of CD10, CD107b, or CD340 markers is low.

[0016] Stem cells exhibiting a novel CD marker expression pattern according to the present invention have superior cell proliferation ability and significantly superior differentiation ability into various tissues compared to other trophoblast-derived stem cells, and thus can be effectively utilized for tissue regeneration treatment, particularly for cartilage regeneration and osteoarthritis treatment.

[0017] Figure 1 is a schematic diagram showing a cross-section of the placenta and the trophoblast tissue details corresponding to the example (CT1) and comparative examples (CT, CT2, CT3) of the present invention.

[0018] Figure 2 is a photograph (×100) showing the cell morphology of subcultured (P1) stem cells derived from the trophoblast layer according to the present invention, observed under a microscope.

[0019] Figure 3 is a diagram showing the colony-forming ability of segmented trophoblast layer-derived stem cells according to the present invention (*** p < 0.001, ** p < 0.05).

[0020] Figure 4 is a diagram showing the group doubling time of the segmented trophoblast layer-derived stem cells according to the present invention.

[0021] Figures 5a to 5c are diagrams showing the results of flow cytometry analysis confirming the surface factor expression characteristics of the segmented trophoblast layer-derived stem cells according to the present invention (*** p < 0.001, ** p < 0.05, ** p < 0.01).

[0022] Figure 6 is a diagram showing the results of confirming the degree of differentiation of the segmented trophoblast layer-derived stem cells according to the present invention into chondrocytes (** p < 0.05, ** p < 0.01).

[0023] Figure 7 is a diagram showing the results of confirming the degree of differentiation of the segmented trophoblast layer-derived stem cells according to the present invention into bone cells (** p < 0.01).

[0024] Figure 8 is a diagram showing the results of confirming the degree of differentiation of segmented trophoblast-derived stem cells according to the present invention into adipocytes (** p < 0.05, ** p < 0.01).

[0025] Figure 9a is a diagram showing the results of confirming the cartilage regeneration effect through Safranin-O staining after administering stem cells (CT1) according to the present invention to an animal model of osteoarthritis.

[0026] Figure 9b is a diagram showing the results of confirming the cartilage protection and regeneration effect in the progression of osteoarthritis through quantification of the cartilage regeneration effect using the Osteoarthritis Research Society International (OARSI) score after administration of stem cells (CT1) according to the present invention to an animal model of osteoarthritis (*** p < 0.001).

[0027] The present invention provides stem cells derived from trophoblast tissues, which have higher expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b and CD273 and lower expression of CD10, CD107b or CD340 markers compared to whole trophoblast-derived stem cells, and a composition for tissue regeneration and a cell therapeutic agent including the same.

[0028] The stem cells of the present invention are novel stem cells having a CD marker expression pattern that is completely different from that of whole trophoblast-derived stem cells, and have excellent proliferation and differentiation capabilities into tissues, so they can be utilized in various tissue regeneration fields.

[0029]

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

[0031] In the present invention, "stem cell" refers to a cell that has the ability to self-replicate and differentiate into two or more different types of cells. Depending on their differentiation potential, stem cells can be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.

[0032] In the present invention, "placenta" refers to a biological tissue created for the fetus during pregnancy, and is a disc-shaped organ weighing 500-600 g, with a diameter of 15-20 cm and a thickness of 2-3 cm. One side of the placenta is in contact with the mother, and the other side is in contact with the fetus, and between them, nutrients and oxygen are transferred between the mother's blood and the fetus' blood vessels. The placenta can be broadly divided into three layers: the amnion, the chorion, and the decidua. The side of the placenta that is attached to the uterine wall is called the maternal side, and the side on the fetal side is called the fetal side, and the umbilical cord protrudes from the center of the fetal side. The maternal surface is divided into 15 to 20 placental lobes by the placental septum formed by the protruding decidua, and inside the placental lobes, some of the villi float in the intervillous space filled with maternal blood, and some are fixed to the placental septum, and fetal blood flows within the villi. Fetal blood comes into contact with maternal blood through the villous surface, and histologically, between the two bloods, the trophoblast and the basement membrane, villous stroma, and fetal capillary wall exist from the maternal blood side, and each plays an important role in the transport of various substances. The placental septum extends from the base of the decidua to the chorionic plate, but is not attached to the chorion and is located in the trophoblast layer.

[0033] The stem cell of the present invention is a stem cell derived from a detailed tissue of a trophoblast, and is characterized by having a higher expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 and a lower expression of a CD10, CD107b, or CD340 marker compared to stem cells derived from the entire trophoblast. In addition, more specifically, the stem cell of the present invention may be characterized by having a higher expression of the CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 markers and a lower expression of the CD10, CD107b, and CD340 markers compared to stem cells derived from the entire trophoblast.

[0034] In addition, the trophoblast-derived stem cell of the present invention is a stem cell exhibiting an XY karyotype, and is characterized by being a stem cell of fetal cell origin with a different karyotype from the entire trophoblast-derived stem cell in which XX and XY karyotypes are mixed.

[0035] That is, the stem cells of the present invention exhibit a completely different CD marker expression pattern compared to trophoblast-derived stem cells according to the existing anatomical classification, and it was confirmed that the stem cells of the present invention exhibit superior proliferation ability and cartilage, bone, or fat cell differentiation ability compared to trophoblast-derived stem cells.

[0036] In the present invention, the trophoblast detailed tissue may be characterized as a continuous trophoblast layer portion with a thickness of 1.5 to 4 mm from the trophoblast portion adjacent to the chorionic plate among the entire trophoblast layer, and preferably a continuous trophoblast layer portion with a thickness of 2 to 3 mm.

[0037] In addition, in the present invention, it is preferable that the trophoblast detailed tissue is a tissue corresponding to the 1 / 10th portion closest to the chorionic plate, when the entire trophoblast layer is divided into 10 equal parts based on the length from the chorionic plate to the peripheral portion of the trophoblast layer. The 1 / 10th portion is a length corresponding to the thickness of 1.5 to 4 mm described above.

[0038] In addition, in the present invention, the trophoblast detailed tissue is characterized in that it does not include tissue derived from the placental septum.

[0039] In the present invention, the stem cells derived from the trophoblast tissue (referred to as “CT1” in the present invention) have superior stem cell proliferation ability and more uniform cartilage, bone, and fat cell differentiation ability compared to the comparative stem cells (referred to as “CT2” in the present invention) obtained from the trophoblast tissue corresponding to about 25% thickness of the entire trophoblast layer based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and the comparative stem cells (referred to as “CT3” in the present invention) obtained from the trophoblast tissue corresponding to about 65% thickness, and have different CD markers and karyotype patterns from the comparative stem cells. That is, in the present invention, it was surprisingly confirmed for the first time that the characteristics of the stem cells obtained are very different depending on the standard for subdividing the entire trophoblast layer based on the length from the chorionic plate to the peripheral part of the trophoblast layer.

[0040] Therefore, the stem cell derived from the trophoblast tissue of the present invention may be a stem cell characterized in that it is obtained through the steps of 1) dividing the entire trophoblast layer into 10 parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and obtaining tissue corresponding to the 1 / 10 part closest to the chorionic plate; and 2) obtaining stem cells from the obtained tissue.

[0041]

[0042] In addition, the present invention provides a cell therapy agent for tissue regeneration or a composition for tissue regeneration comprising stem cells derived from the trophoblast sub-tissue.

[0043] The stem cells derived from the trophoblast tissue of the present invention not only exhibit excellent proliferative capacity, but can also differentiate into various types of cells such as fat cells, chondrocytes, bone cells, nerve cells, ligament cells, or tenocytes, and therefore can be utilized as a cell therapeutic agent for the purpose of tissue regeneration and a medical device that is a composition for tissue regeneration.

[0044] Stem cells included in the tissue regeneration composition of the present invention may include, without limitation, allogeneic, syngeneic, or xenogeneic origin.

[0045] In the present invention, the term "cellular therapeutic agent" refers to a medicine (as defined by the US FDA) used for the purposes of treatment, diagnosis, and prevention by cells and tissues isolated, cultured, and manufactured through special manipulation from humans, and refers to a medicine used for the purposes of treatment, diagnosis, and prevention of diseases by a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods to restore the function of cells or tissues.

[0046] The stem cells of the present invention are stem cells with excellent differentiation potential that can differentiate into cartilage, bone, fat, etc., and thus, by administering them to joints, cartilage, tendons, or ligaments, they can treat or prevent various lesions, such as lesions of articular cartilage, and when administered intra-articularly, they can regenerate the cartilage surface to a smooth and almost damage-free state, thereby protecting and regenerating cartilage to prevent the progression of arthritis.

[0047] In the present invention, "differentiation" generally refers to a phenomenon in which a relatively simple boundary separates into two or more qualitatively different subsystems. Specifically, it refers to a phenomenon in which cells become specialized in structure or function while growing through division and proliferation, i.e., a phenomenon in which cells, tissues, etc. of a living organism change in form or function to perform their respective assigned tasks. Comparatively, "undifferentiated" refers to a state in which the aforementioned differentiation has not occurred and still contains the characteristics of a stem cell.

[0048] The method for differentiating stem cells can be performed according to a conventionally known method and is not particularly limited. For example, it is preferable to use a method for differentiating the stem cells into adipocytes by culturing them in a medium containing dexamethasone, indomethacin, insulin, and IBMX (3-isobutyl-1-methylxanthine); a method for differentiating the stem cells into chondrocytes by culturing them in a medium containing dexamethasone, bone morphogenetic protein 6 (BMP-6), transforming growth factor beta (TGF-β), ascorbic acid, and L-proline; a method for differentiating the stem cells into osteocytes by culturing them in a medium containing dexamethasone, ascorbic acid, β-glycrophosphate, and ascorbic acid-2-phosphate.

[0049] The method for measuring the degree of differentiation of stem cells differentiated by the above method is not particularly limited thereto, but may use techniques known in the art, such as flow cytometry, immunocytochemical methods, methods for measuring changes in cell surface markers or morphology using gene-expression profiles, methods for examining changes in cell morphology using an optical microscope or a confocal microscope, etc., and preferably, Oil-red O staining, GAG amount measurement, or Calcium amount measurement, etc. may be used.

[0050] The stem cells according to the present invention exhibit homogeneous growth characteristics and differentiation characteristics compared to conventional stem cells derived from the entire tissue of the placental trophoblast layer, and have an excellent tissue regeneration effect in an animal model of osteoarthritis disease.

[0051] Preferably, the tissue regeneration composition and cell therapy agent of the present invention can be used for the purpose of regenerating one or more tissues selected from the group consisting of fat, cartilage, bone, nerve, ligament, and tendon.

[0052] In addition, in the present invention, the cartilage may include, without limitation, hyaline cartilage, fibrocartilage, or elastic cartilage, and the cartilage may be characterized in that it is selected from the group consisting of articular cartilage, ear cartilage, nasal cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, coronal cartilage, laryngeal cartilage, and spinal cartilage.

[0053] The above fat includes all fat regardless of location in the body, and includes, but is not limited to, subcutaneous fat, fat located between the stomach and intestines (omentum, mesentery), bone marrow fat, and retroperitoneal fat.

[0054] Therefore, the tissue regeneration composition and cell therapy agent of the present invention can be used for treating cartilage damage, cartilage defect, bone defect, tendon-ligament defect, adipose tissue defect, cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma, arthritis, cartilage deficiency, or congenital tracheal softening.

[0055] In the present invention, “cartilage defect” is a comprehensive meaning that includes cases where there is damage, defect, or deficiency in cartilage contained in the body, and includes, but is not limited to, cartilage trauma, cartilage rupture, cartilage softening, cartilage necrosis, osteochondritis, cartilage defect, or osteoarthritis.

[0056] In addition, the cell therapy agent or tissue regeneration composition of the present invention can be administered directly into a joint or joint cavity, and can be used to treat lesions of articular cartilage, or for therapeutic or preventive purposes by administering it to a tendon or ligament area. For example, by administering the stem cells of the present invention to a joint, tendon, or ligament area, it can be used to promote recovery or adjustment of damaged areas of the tissues, or it can be used to treat joint tissues (e.g., knee joints, etc.) by reconstructing or regenerating them using stem cell-derived materials, such as cartilage tissue components derived from the stem cells of the present invention.

[0057] The stem cells of the present invention can be administered in the form of an injection and may further include a hydrogel.

[0058] The hydrogel that can be used in the cell therapeutic agent of the present invention may include, without limitation, hydrogels known in the art suitable for injection, and may be at least one selected from the group consisting of small intestinal submucosal tissue, hyaluronic acid, carboxymethylcellulose (CMC), alginate, chitosan, polyacrylamide, poly(N-isopropylacrylamide), β-glycerophosphate, poly(ethylene oxide)poly(propylene oxide)poly(ethyleneoxide), Pluronic, and a mixture of carboxymethylcellulose (CMC) and polyethyleneimine (PEI).

[0059] The tissue regeneration composition described above can be administered to individuals, including humans, and preferably, can be administered to mammals, including humans, without limitation. For example, mammals to which the tissue regeneration composition of the present invention can be administered include companion animals such as dogs and cats, and livestock animals such as horses, cows, and pigs, and the desired effects can be equally achieved by administration to these animals.

[0060] Therefore, the tissue regeneration composition of the present invention may be a veterinary composition. Furthermore, if the tissue regeneration composition is a veterinary composition, the stem cells may be of xenogeneic origin, for example, stem cells isolated from humans may be administered to non-human animals for veterinary purposes.

[0061] The preferred dosage of the cell therapy agent of the present invention varies depending on the individual's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration of the treatment, but can be appropriately selected by those skilled in the art. Administration may be administered once daily or in multiple divided doses. The above dosage does not limit the scope of the present invention in any way.

[0062]

[0063] In addition, the present invention provides a method for obtaining stem cells, comprising the steps of: 1) dividing the entire trophoblast layer into 10 parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and obtaining tissue corresponding to the 1 / 10 part closest to the chorionic plate; and 2) obtaining stem cells from the obtained tissue; wherein the stem cells are trophoblast-derived tissue-derived stem cells that have higher expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 and lower expression of CD10, CD107b, or CD340 markers compared to entire trophoblast-derived stem cells.

[0064] The above step 2) can be performed by performing an enzymatic reaction by adding an enzyme solution to trophoblast tissue separated from the placenta, culturing the obtained cells in a medium supplemented with fetal bovine serum and antibiotics without using growth factors, and then recovering the cells. The enzymes include, but are not limited to, trypsin, collagenase, dispase, DNase, RNase, protease, lipase, hyaluronidase, and elastase. The collagenase may include collagenase A, I, II, III, or IV.

[0065] The stem cell according to the present invention is characterized by having morphological characteristics of a fibroblast shape, excellent proliferation ability, differentiation ability into fat, cartilage or bone cells, and colony-forming ability, and is characterized by having high expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b and CD273 compared to the whole medullary membrane-derived stem cell, and low expression of CD10, CD107b or CD340 marker, and preferably, high expression of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b and CD273 marker, and low expression of CD10, CD107b and CD340 marker, and is characterized by having CD29, CD44, CD49a, CD49d, CD71, CD73, CD81, CD90, CD105 It may exhibit a positive immunological characteristic with at least 50% expression of CD108, CD140b, CD166, and CD273; and a negative immunological characteristic with at most 10%, preferably at most 5%, expression of CD11b, CD14, CD19, CD34, CD45, CD10, CD107b, and CD340.

[0066]

[0067] In addition, the present invention provides a method for producing a composition for tissue regeneration, which further includes a step of mixing the obtained stem cells with a hydrogel in the above obtaining method.

[0068]

[0069] In addition, the present invention provides a tissue regeneration method comprising a step of administering stem cells derived from trophoblast sub-tissue to an individual in need thereof, wherein the stem cells derived from trophoblast sub-tissue are characterized in that, compared to whole trophoblast-derived stem cells, the expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 is high, and the expression of CD10, CD107b, or CD340 markers is low.

[0070] When used in the above tissue regeneration method, the stem cells may be of allogeneic origin, syngeneic origin, or xenogeneic origin, and may be characterized by administering allogeneic or xenogeneic stem cells to an individual in need thereof.

[0071] Furthermore, when the stem cells of the present invention are used for tissue regeneration purposes, they may be administered alone or in a mixture of stem cells and hydrogel. The hydrogels that can be used in tissue regeneration methods may utilize the same compositions described above for tissue regeneration compositions or cell therapy agents, and are omitted to avoid overly complex descriptions.

[0072] In addition, the subject to which the tissue regeneration method of the present invention is applied may be a human or a non-human mammal, and in the case of a non-human mammal, it may be used for veterinary purposes and may be administered without limitation to companion animals such as dogs and cats and livestock animals such as horses, cows, and pigs.

[0073] When the tissue regeneration method of the present invention is used for veterinary purposes, the stem cells may be of xenogeneic origin, for example, stem cells isolated from humans may be administered to non-human animals for veterinary purposes.

[0074] The target tissue to which the method of the present invention can be applied can utilize the same composition as described above for the tissue regeneration composition or cell therapy agent, and is omitted to avoid complexity in the description.

[0075]

[0076] 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.

[0077] Example 1.

[0078] 1. Obtaining stem cells derived from the trophoblast layer, a detailed tissue of the placenta.

[0079] Placentas were collected from mothers who consented to donation after normal delivery by cesarean section at Chung-Ang University Gwangmyeong Hospital in accordance with the guidelines of the Institutional Review Board (IRB) of the Public Interest Committee. The collected placentas were transferred into sterile containers, and after stripping the amniotic membrane from the placental tissue, the chorion and the trophoblast tissue, approximately 10% of the thickness of the area adjacent to the chorion, were separated using sterile scissors. That is, when the entire separated trophoblast layer is divided into 10 equal parts, the area corresponding to approximately 1 / 10 of the point closest to the chorion was obtained. In other words, the entire trophoblast layer was divided into 10 equal parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and the area corresponding to the 1 / 10th area closest to the chorionic plate was cut to obtain the tissue. The cut area also includes the cut portion of the villi that were separated together. The separated trophoblast tissue was transferred to a 150 mm dish, the chorion was removed using a scalpel, and blood and blood cells were removed by washing more than 5 times using PBS.

[0080] After transferring the washed trophoblast tissue to a 50 ml tube, α-MEM medium supplemented with 0.2% collagenase was added and reacted for 2-3 hours using a shaker at 37℃ to obtain cells derived from the trophoblast layer. The cells derived from the obtained trophoblast layer were filtered through a 100 ㎛ mesh to remove undigested tissue, and α-MEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25℃ and 1200 rpm for 4 minutes. The supernatant was removed, and α-MEM medium supplemented with fetal bovine serum and antibiotics but not containing growth factors was added to the remaining precipitated cells, and cultured under 37℃ and 5% CO2 conditions. Cells attached to the bottom of the culture vessel were selected from the above culture to obtain stem cells derived from the detailed trophoblast layer. Stem cells obtained from the trophoblast tissue, which does not include the placental septum, corresponding to 10% of the thickness of the soft part of the chorion, were referred to as “CT1.”

[0081]

[0082] Comparative Example 1: Obtaining stem cells derived from trophoblast subtissue and whole tissue

[0083] After stripping the amniotic membrane from the placental tissue, the entire trophoblast tissue, including the trophoblast tissue and decidua, which are continuous from the chorion to about 25% thickness and about 65% thickness, were dissected, transferred to a 150 mm dish, and the chorion was removed using a scalpel. Afterwards, the separated tissues were washed with PBS to remove blood and blood cells from the placental tissue. In addition, to obtain the entire trophoblast tissue, the chorion was removed using a scalpel, and the decidua was removed using sterilized scissors. The remaining entire trophoblast tissue was transferred to a new 150 mm dish, washed with PBS, and blood and blood cells were removed from the placental tissue. The washed tissue was added to a-MEM medium supplemented with 0.2% collagenase, and reacted using a shaker at 37°C to obtain placental cells. The obtained placental cells were filtered through a 100 ㎛ mesh to remove undigested tissue, and DMEM medium supplemented with fetal bovine serum and antibiotics was added, followed by centrifugation at 25°C and 1200 rpm for 4 minutes. The supernatant was removed, and a-MEM medium supplemented with fetal bovine serum and antibiotics but not containing growth factors was added to the remaining precipitated cells, and cultured at 37°C under 5% CO2 conditions. Cells attached to the bottom of the culture vessel were selected from the above culture, and stem cells obtained from the trophoblast tissue corresponding to approximately 25% of the thickness of the continuous area from the chorion were designated as “CT2”, stem cells obtained from the trophoblast tissue corresponding to approximately 65% ​​of the thickness of the soft area of ​​the chorion were designated as “CT3”, and stem cells obtained from the entire trophoblast layer were designated as “CT”.

[0084] The obtaining site of stem cells according to the present invention and stem cells obtained in comparative examples is schematically shown in Fig. 1.

[0085]

[0086] Example 2. Subculture of stem cells derived from the trophoblast layer, a detailed tissue of the placenta.

[0087] The stem cells obtained in Example 1 were washed with PBS and then cultured in α-MEM medium containing fetal bovine serum and antibiotics but without growth factors, with the medium replaced every 2 to 3 days. When the stem cells grew 70 to 80% or more, the stem cells were treated with TryPLE to isolate the stem cells from the culture vessel, diluted to a ratio of 1 / 4, and cultured in another culture vessel to perform subculture. The cell morphology after culture was observed under a microscope at passage 1. In addition, using the CT2, CT3, and CT-derived stem cells obtained in Comparative Example 1, subculture was performed using the same method, and the cell morphology after culture was observed under a microscope. The results are shown in Fig. 2.

[0088] As shown in Fig. 2, it was confirmed that all obtained stem cells exhibited morphological characteristics similar to fibroblasts.

[0089]

[0090] Example 3. Analysis of colony-forming capacity of stem cells derived from the trophoblast layer, a detailed tissue of the placenta.

[0091] The colony-forming ability of stem cells derived from the trophoblast tissue obtained in Example 1 was confirmed. More specifically, the stem cells derived from the trophoblast layer obtained in Example 1 were subjected to the first subculture using the method of Example 2, and at the end of the subculture, 5 × 10 were seeded in a 100 mm dish. 3After seeding each stem cell, they were cultured for 10 days in α-MEM medium supplemented with fetal bovine serum and antibiotics but without growth factors. The cultured stem cells were subjected to Giemsa staining to count the number of colonies formed from the stem cells. In addition, colony forming ability was measured using the CT2, CT3, and CT-derived stem cells obtained in Comparative Example 1 using the same method. In the case of colony forming ability, the result value of CT-derived stem cells was converted to 100%. The results are shown in Fig. 3.

[0092] As shown in Fig. 3, the stem cells derived from the trophoblast layer according to the present invention exhibited colony-forming ability that was approximately 1.7 times better than that of the entire trophoblast-derived stem cells, and were confirmed to exhibit colony-forming ability that was better than that of CT2 and CT3-derived stem cells.

[0093]

[0094] Example 4. Analysis of the population doubling time of stem cells derived from the trophoblast layer, a detailed tissue of the placenta.

[0095] The population doubling time (PDT) of the stem cells derived from the trophoblast tissue obtained in Example 1 was confirmed. More specifically, the stem cells from Example 1 were cultured for the first passage using the method of Example 2, and in the 2nd to 6th passage culture stages, 3 × 10 cells were cultured in a 100 mm dish. 5After seeding each stem cell, the number of stem cells was counted when the growth was 80-90% or more. The population doubling time of stem cells was calculated for the 2nd to 6th passage culture using the following formulas: GR = (N f―Ni) / (CT X 100cm2), PD = log(N f / Ni) / log2, PDT = CT / PD (Nf, final cell number; Ni, initial cell number; PD, population doubling; CT, culture time). The results of confirming the population doubling time of each stem cell are shown in Fig. 4.

[0096] As shown in Fig. 4, the CT1-derived stem cells according to the present invention exhibited shorter population doubling times than the CT and CT3-derived stem cells at all passages, and compared to the CT2-derived stem cells, the population doubling times were also confirmed to be shorter at almost all passages. This result demonstrates that the CT1-derived stem cells of the present invention have excellent proliferative capacity and can proliferate rapidly in a short period of time.

[0097]

[0098] Example 5. Confirmation of the cell origin of stem cells derived from the trophoblast layer, a detailed tissue of the placenta.

[0099] The placenta is a special organ in which cells of maternal and fetal origin coexist, and both maternal and fetal cells exist in the placenta. Accordingly, an experiment was performed to confirm the cell origin of the CT1 cells obtained in Example 1 and the cells of Comparative Example 1. Cells obtained at the initial culture stage P2 and P5, which are known to be widely applied in clinical practice, were spread on slides and stained using X and Y chromosome probes labeled with Cy3 (red) and fluorescein isothiocyanate (green), respectively (Vysis, Downers Grove, IL, USA). After staining the cell nucleus with DAPI, the slide analysis was performed using spectral red and green filters under ×1000 magnification, and the X and Y chromosomes expressed in the cells were counted and calculated as a %, and the results are shown in Table 1.

[0100]

[0101]

[0102] As shown in Table 1, in the case of CT, it was confirmed that XX and XY were mixed in P2 and P5, and in P5, CT1 was confirmed to be XY, CT2 and CT3 were confirmed to be XX. Through the above results, it was confirmed that CT1, unlike CT2 and CT3, exhibits fetal stem cell characteristics.

[0103]

[0104] Example 6. Surface marker analysis of stem cells derived from the trophoblast layer, a detailed tissue of the placenta.

[0105] To confirm the immunological characteristics of the trophoblast-derived stem cells obtained in Example 1, the following experiment was performed using 242 Human Cell Surface Marker Screening Panels. First, stem cells derived from the trophoblast layer were washed with PBS, triple-treated, and then harvested and centrifuged at 1200 rpm for 4 minutes. After removing the supernatant, the stem cells were suspended in PBS and passed through a 40 μm cell strainer. The cells and BD Pharmingen Stain Buffer + EDTA were prepared and mixed. 100 μl was dispensed into each well of a Falcon® round-bottom 96-well plate (Cat. 353910) (100,000 cells / well). Then, 20 μl of the corresponding antibody was dispensed 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. The supernatant was carefully removed, washed with BD Pharmingen Stain Buffer + EDTA, and centrifuged at 1200 rpm for 5 minutes. 100 μl of secondary antibody was then dispensed into each well. BD Pharmingen Stain Buffer + EDTA was added to each well for washing, centrifuged at 1200 rpm for 5 minutes, the supernatant was removed, and the washing process was carefully repeated twice with 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 immunological characteristics of CT2, CT3 and CT-derived stem cells obtained in Comparative Example 1 were analyzed using the same method, and common stem cell surface markers are shown in Table 2, and after analyzing 242 surface markers, markers showing significant differences in expression patterns in each stem cell are shown in Figure 5.

[0106]

[0107] As shown in Table 2, CT, CT1, CT2, and CT3-derived stem cells were confirmed to exhibit positive expression characteristics for CD44, CD73, CD90, CD105, and CD166, and negative expression characteristics for CD11b, CD14, CD19, CD34, CD45, and HLA-DR.

[0108] In addition, as shown in FIGS. 5A to 5C, the CT1-derived stem cells according to the present invention exhibited a unique CD marker expression pattern that was distinct from the CT, CT2, and CT3-derived stem cells that exhibited similar CD marker expression patterns. Specifically, it was confirmed that the CT1-derived stem cells according to the present invention had a high expression rate for the CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 markers, and a low expression rate for the CD10, CD107b, and CD340 markers.

[0109] Through these results, it was confirmed that the CT1-derived stem cells of the present invention are stem cells having a novel CD marker expression pattern that is distinct from other trophoblast-derived stem cells.

[0110]

[0111] Example 7. Confirmation of the differentiation potential of stem cells derived from the trophoblast layer, a detailed tissue of the placenta, into chondrocytes.

[0112] In order to confirm the differentiation ability of CT1-derived stem cells obtained in Example 1 into chondrocytes, the stem cells were cultured in a known chondrocyte differentiation inducing medium (a-MEM medium containing 0.1 μM dexamethasone, 50 μg / mL ascorbic acid, 40 μg / mL L-proline, 10 ng / mL TGF-β3, 500 ng / mL BMP-6, and 50 mg / mL ITS premix) for 3 weeks to induce differentiation into chondrocytes. In order to measure the degree of differentiation of the stem cells into chondrocytes, the sulfated glycosaminoglycan (GAG) content was measured using a Glycan sulfated GAG assay kit (Biocolor Ltd., Carrickfergus, County Antrim, UK). The absorbance was measured in a 96-well plate at a wavelength of 656 nm using a microplate reader. In addition, the differentiation potential of CT-derived stem cells obtained in Comparative Example 1 into chondrocytes was measured using the same method. The results are shown in Fig. 6.

[0113] As shown in Fig. 6, it was confirmed that the trophoblast layer-derived stem cells according to the present invention have superior chondrocyte differentiation ability that can be differentiated into chondrocytes more uniformly than CT-derived stem cells.

[0114]

[0115] Example 8: Confirmation of the differentiation potential of stem cells derived from the trophoblast layer, a detailed tissue of the placenta, into osteocytes.

[0116] In order to confirm the differentiation ability of CT1-derived stem cells obtained in Example 1 into osteocytes, the stem cells were cultured in a known osteocyte differentiation inducing medium (a-MEM medium containing 10% FBS, 1% antibiotics, 100 μM dexamethasone, 50 mM ascorbic acid-2-phosphate, 10 μM β-glycophosphate, and 250 μM ascorbic acid) for 4 weeks to induce differentiation into osteocytes. After 4 weeks, the cells were treated with 0.6 M HCl for 24 hours and stored at 37°C to measure intracellular calcium. The absorbance was measured at 565 nm using the o-cresolphthalein complexone method (Pointe Scientific, Canton, MI, USA), and the calcium concentration was standardized and quantified.

[0117] Additionally, the differentiation potential of CT-derived stem cells obtained in Comparative Example 1 into bone cells was measured using the same method. The results are shown in Fig. 7.

[0118] As shown in Fig. 7, it was confirmed that the trophoblast layer-derived stem cells according to the present invention have superior osteocyte differentiation ability that can be differentiated into osteocytes more uniformly than CT-derived stem cells.

[0119]

[0120] Example 8: Confirmation of the ability of stem cells derived from the trophoblast layer, a detailed tissue of the placenta, to differentiate into adipocytes.

[0121] In order to confirm the differentiation ability of CT1-derived stem cells obtained in Example 1 into adipocytes, the stem cells were cultured for 3 weeks by alternately adding known adipocyte differentiation induction medium 1 (a-MEM medium containing 10% FBS, 1% Antibiotics, 1 μM dexamethasone, 20 μM indomethacin, 10 μM insulin, and 50 μM 3-isobutyl-1-methylxanthine (IBMX)) and adipocyte differentiation induction medium 2 (DMEM medium containing 10% FBS, 1% Antibiotics, and 10 μM insulin) every 3 to 4 days to induce differentiation into adipocytes. In order to measure the degree of differentiation of the stem cells into adipocytes, after Oil red O staining according to a conventionally known method, lipids were dissolved by treating with a 100% isopropanol solution, and the absorbance at 500 nm was measured. Additionally, the differentiation potential of CT-derived stem cells obtained in Comparative Example 1 into adipocytes was measured using the same method. The results are shown in Fig. 8.

[0122] As shown in Fig. 8, it was confirmed that the trophoblast layer-derived stem cells according to the present invention have superior adipocyte differentiation ability that can be differentiated into adipocytes more uniformly than CT-derived stem cells.

[0123]

[0124] Example 9: Verification of the efficacy of stem cells derived from the trophoblast layer, a detailed tissue of the placenta, as a cell therapy in an animal model of osteoarthritis.

[0125] In order to verify the effectiveness of the CT1-derived stem cells obtained in Example 1 as a cell therapy agent, the following experiments were performed using an osteoarthritis animal model. More specifically, in order to create an osteoarthritis animal model in a Wistar rat (male, 12 weeks old), a healthy rat was selected and anesthetized by injection with an appropriate amount of ketamine and lumpun according to the body weight. After confirming that the rat was sufficiently anesthetized, the knee joint area of ​​both lower extremities was shaved and fixed with a bandage while maintaining the posture. The knee joint area on both sides was disinfected with povidone, and the patella was palpated to confirm the position. Then, a paramedian approach was made along an incision line passing above, below, and on the medial side of the patella to reach the inside of the knee joint, and the patella was flexed while being pulled outward to observe the inside of the joint. After confirming that there were no specific pathological findings, a destabilization of the medial meniscus (DMM) model was created by injuring the meniscus. Eight weeks after inducing the injury as described above, 20 μl of 1 ml of CT1 stem cell suspension mixed with 4% hyaluronic acid was injected into the joint space of the animal model using a syringe. After confirming that the rats woke up from anesthesia, they were allowed to move freely, and analgesics and antibiotics were administered for 3 days after surgery to prevent infection. After 4 and 8 weeks, sections of the injured and treated joint areas were obtained from each rat, and Safranin O staining was performed, and the degree of newly formed cartilage regeneration was analyzed by quantification using the OARSI score. The results are shown in Figures 9a and 9b, respectively.

[0126] As shown in Fig. 9a, 4 and 8 weeks after injection of CT1 stem cells, it was confirmed that the cartilage surface was smoother and regenerated with almost no cartilage damage compared to the saline-injected control group. In addition, as shown in Fig. 9b, the OARSI score results showed that the group injected with CT1 stem cells (2.1±0.7) had a lower score than the control group (7.2±0.4) after 4 weeks, and that the group injected with CT1 stem cells (2.0±0.8) had a lower score than the control group (9.2±0.5) at 8 weeks. Therefore, it was confirmed that CT1 stem cells play a role in protecting and regenerating cartilage to prevent further progression of arthritis during the progression of osteoarthritis.

[0127]

[0128] Through the above experimental results, it was confirmed that the stem cells derived from the trophoblast layer, a detailed tissue of the placenta according to the present invention, are stem cells that exhibit a specific CD marker expression pattern, exhibit excellent differentiation and proliferation capabilities, and exhibit superior arthritis treatment effects compared to conventional stem cells derived from the entire placental trophoblast layer. In particular, the stem cells derived from the trophoblast layer according to the present invention showed consistent patterns in the characteristics of growth, proliferation, morphology, and differentiation. Therefore, it was confirmed that when stem cells derived from the trophoblast layer are used, the differentiation efficiency into target cells can be improved, and that they can be usefully used as cell therapy for various diseases.

Claims

1. Compared to whole trophoblast-derived stem cells High expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273; Stem cells derived from trophoblastic tissue with low expression of CD10, CD107b or CD340 markers.

2. In paragraph 1, the stem cell Compared to whole-cell membrane-derived stem cells, Stem cells characterized by high expression of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273 markers and low expression of CD10, CD107b, and CD340 markers.

3. A stem cell according to claim 1, characterized in that the stem cell is a stem cell of fetal cell origin.

4. In the first paragraph, the stem cell is a stem cell having cartilage, bone, or fat differentiation potential.

5. In the first paragraph, the trophoblast detailed tissue is a stem cell that is a continuous trophoblast layer portion with a thickness of 1.5 to 4 mm from the trophoblast portion adjacent to the chorionic plate among the entire trophoblast layer.

6. In the fifth paragraph, the trophoblast detailed tissue is a stem cell, which is a tissue corresponding to the 1 / 10th part closest to the chorionic plate, when the entire trophoblast layer is divided into 10 parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer.

7. Stem cell according to claim 5, wherein the amniotic membrane tissue does not include tissue derived from the placental septum.

8. In paragraph 1, the stem cell 1) A step of dividing the entire trophoblast layer into 10 equal parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and obtaining tissue corresponding to the 1 / 10th part closest to the chorionic plate; and 2) A stem cell obtained through a step of obtaining stem cells from the obtained tissue; 9. A composition for tissue regeneration comprising the stem cell of any one of claims 1 to 8 as an active ingredient.

10. A composition for tissue regeneration in claim 9, wherein the tissue is at least one selected from the group consisting of fat, cartilage, bone, nerve, ligament, and tendon.

11. A composition for tissue regeneration, characterized in that the cartilage in claim 10 is hyaline cartilage, fibrocartilage or elastic cartilage.

12. A composition for tissue regeneration, characterized in that in claim 10, the cartilage is selected from the group consisting of articular cartilage, ear cartilage, nasal cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, coronal cartilage, laryngeal cartilage, and spinal cartilage.

13. A composition for tissue regeneration, characterized in that the composition in claim 9 is for the treatment of cartilage damage, cartilage defect, bone defect, tendon-ligament defect, adipose tissue defect, cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma, arthritis, cartilage deficiency, or congenital tracheal softening.

14. A tissue regeneration composition according to claim 9, characterized in that the tissue regeneration composition is an injection.

15. A composition for tissue regeneration according to claim 14, characterized in that the composition for tissue regeneration further comprises a hydrogel.

16. A composition according to claim 9, wherein the tissue regeneration composition is a veterinary composition.

17. A cell therapeutic agent for tissue regeneration comprising a stem cell of any one of claims 1 to 8. 18.1) A step of dividing the entire trophoblast layer into 10 equal parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and obtaining tissue corresponding to the 1 / 10th part closest to the chorionic plate; and 2) A method for obtaining stem cells, including a step of obtaining stem cells from the obtained tissue; The above stem cells are compared to the stem cells derived from the whole mesenchyme. High expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273; A method for obtaining stem cells derived from trophoblast tissue, which are stem cells with low expression of CD10, CD107b or CD340 markers. 19.1) A step of dividing the entire trophoblast layer into 10 equal parts based on the length from the chorionic plate to the peripheral part of the trophoblast layer, and obtaining tissue corresponding to the 1 / 10th part closest to the chorionic plate; 2) A step of obtaining stem cells from the obtained tissue; and 3) A method for producing a composition for tissue regeneration, comprising: a step of mixing the obtained stem cells with a hydrogel; The above stem cells are compared to the stem cells derived from the whole mesenchyme. High expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273; A method for producing a composition for tissue regeneration, the composition being stem cells with low expression of CD10, CD107b or CD340 markers.

20. A method for tissue regeneration, comprising the step of administering stem cells derived from trophoblast tissue to an individual in need thereof, The above trophoblast-derived stem cells are Compared to whole trophoblast-derived stem cells, High expression of two or more markers selected from the group consisting of CD29, CD49a, CD49d, CD71, CD81, CD108, CD140b, and CD273; A method for tissue regeneration characterized by low expression of CD10, CD107b or CD340 markers.

21. A method for tissue regeneration in claim 20, wherein the stem cells are of homologous or xenogeneic origin.

22. A method for tissue regeneration in claim 20, wherein the trophoblast-derived tissue-derived stem cells are administered together with a hydrogel.

23. A method for tissue regeneration in claim 20, wherein the subject is a human or non-human mammal.

Images