KR100871984B1 - Multipotent Stem Cell Derived from Placenta Tissue and Cellular Therapeutic Agents Comprising the Same - Google Patents

Abstract

The present invention relates to a placental tissue-derived pluripotent stem cell and a cell therapeutic agent containing the same, and more particularly, characterized in that the fine amnion, membrane, basal degeneration membrane or placental tissue is cultured in bFGF-containing medium and then recovered. (A) all showed positive immunological properties for CD29, CD44, CD73, CD90 and CD105, and all negative immunological properties for CD31, CD34, CD45 and HLA-DR; (b) exhibits positive immunological properties for Oct4 and SSEA4; (c) attached to plastic and grown, exhibits morphological characteristics of round- or spindle-shape, and forms a sphere in SFM medium (sphere-forming medium) to form micronized Long-term maintenance; And (d) a method for producing placental stem cells having the ability to differentiate into mesoderm, endoderm and ectoderm derived cells, and obtained placental stem cells. Pluripotent stem cells according to the present invention have the ability to differentiate into myocytes, vascular endothelial cells, osteoblasts, neurons, astrocytes, adipocytes, chondrocytes, osteoblasts, or insulin secreting pancreatic beta cells, It is effective in the treatment of muscle diseases, osteoporosis, osteoarthritis, neurological diseases, diabetes and the like, and useful for forming breast tissue.

Placental tissue, placental stem cells, differentiation, cell therapy

Description

Multipotent Stem Cell Derived from Placenta Tissue and Cellular Therapeutic Agents Comprising the Same}

1 is a micrograph of the morphology of placental mesenchymal stem cells (MSCs).

Figure 2 shows the results of analyzing the surface antigens of basal decidual membrane-derived MSC using flow cytometry (FACS).

Figure 3 shows the results of analyzing the surface antigens of the amniotic membrane-derived MSC using flow cytometry (FACS).

FIG. 4A is a sphere formation photograph of the first passage amniotic membrane cultured in SFM medium for 3 days, and B is a sphere formation photograph of the first passage basal drop membrane cultured in SFM medium for 7 days.

FIG. 5 shows immunohistochemistry for amnion (A) and basal degeneration membrane (B) derived stem cells induced in MM-3160 medium (SKBM medium for muscle cell differentiation) for 10 days in myogenesis. (αMyosin-FITC) is a micrograph

FIG. 6 shows immunohistochemistry for stem cells derived from amnion (A) and basal shedding membrane (B) induced in DMEM medium and NM3229 medium (neural cell differentiation medium) for 10 days in neuronal differentiation. Micrograph of the test (αGFAP-FITC).

7 is a photograph stained by Alizarin red S staining for stem cells derived from amnion (B) and basal decidual membrane (C) in osteoblast differentiation (Osteogenesis) [A: control group; B: amnion derived stem cells under osteoblast differentiation conditions; C: basal decidual stem cells under osteoblast differentiation conditions].

FIG. 8 is a photograph stained by oil red O staining of stem cells derived from amnion (B) and basal decidual membrane (A) in adipogenesis.

9 shows RT-PCR results for OCT4 [lane 1: marker; Lane 2: RT-reaction control; Lane 3: amnion stem cells; Lane 4: basal decidual stem cells; Lane 5: PCR-reaction control].

10 is a photograph of immunohistochemistry using specific antibodies indicated in each [A: OCT4; B: SSEA4; C: CD44, CD54 and control.

FIG. 11 is a graph showing cumulative population doubling level (CPDL) according to passage number for basal decidual-derived stem cells. FIG.

12 is a graph showing accumulation group doubling degree (CPDL) according to passage number for amnion derived stem cells.

The present invention relates to a placental tissue-derived pluripotent stem cells and cell therapies containing the same, the present invention (a) CD29, CD44, CD73, CD90 and CD105 all show positive immunological properties, CD31, CD34, Both show negative immunological properties for CD45 and HLA-DR; (b) exhibits positive immunological properties for Oct4 and SSEA4; (c) adhered to plastic and grown, exhibit morphological properties of round- or spine-shape, and form spheres in SFM media to allow long-term maintenance in undifferentiated state; And (d) placental stem cells exhibiting the ability to differentiate into mesoderm, endoderm and ectoderm derived cells.

Biotechnology in the 21st century presents the possibility of new solutions to food, environment and health problems as the final goal of human welfare. Recently, the use of stem cells has emerged as a new chapter in the treatment of intractable diseases. Previously, organ transplantation and gene therapy have been suggested for the treatment of intractable disease in humans. However, due to the lack of immunity, lack of supply organs, lack of knowledge on vector development and disease genes, efficient practical use has been insufficient.

As interest in stem cell research increased, pluripotent stem cells with the ability to form all organs through proliferation and differentiation were found to be capable of fundamentally solving long-term damage as well as treating most diseases. In addition, many scientists have suggested the possibility of applying stem cells to treatment of almost all organs of the human body as well as treatment of Parkinson's disease, various cancers, diabetes and spinal cord injury.

Stem cells are cells that have the ability to self-replicate and differentiate into two or more cells, totipotent stem cells, pluripotent stem cells, and c. It can be classified as multipotent stem cells.

Pluripotent stem cells are pluripotent cells that can develop into a complete individual. Cells up to the eighth cell after fertilization of eggs and sperm have this property. Transplantation can result in one complete individual. Pluripotent stem cells are cells that can develop into a variety of cells and tissues derived from ectoderm, mesoderm, and endodermal layer. The inner cell mass located inside the blastocyst after 4-5 days of fertilization. It is called embryonic stem cells and differentiates into a variety of other tissue cells but does not form new life. Pluripotent (multipotent) stem cells are stem cells that can only differentiate into specific cells that form the tissues and organs that contain them.

Pluripotent stem cells were first isolated from adult bone marrow (Y. Jiang et al., Nature , 418: 41, 2002) and subsequently identified in other adult tissues (CM Verfaillie, Trends Cell Biol. , 12: 502, 2002). In other words, bone marrow is the most widely known source of stem cells, but pluripotent stem cells have also been identified from skin, blood vessels, muscle and brain (JG Toma et al., Nat. Cell Biol ., 3: 778, 2001; M Sampaolesi et al, Science , 301: 487, 2003; Y. Jiang et al., Exp. Hematol. , 30: 896, 2002). However, stem cells in adult tissues such as bone marrow are very rare, and these cells are difficult to culture without induction of differentiation, making them difficult to culture without specifically screened media. That is, it is very difficult to separate stem cells and preserve them in vitro.

In response, it has recently been found that adipose tissue is a new source of pluripotent stem cells (B. Cousin et al., BBRC ., 301: 1016, 2003; A. Miranville et al., Circulation, 110: 349, 2004; S. Gronthos et al., J. Cell Physiol ., 189: 54, 2001; MJSeo et al., BBRC ., 328: 258, 2005). In other words, it has been reported that human adipose tissue obtained by liposuction contains undifferentiated cell populations, which are cells that have differentiation ability into adipocytes, osteoblasts, myoblasts and chondrocytes in vitro (PAZuk). et al., Tissue Eng. , 7: 211, 2001; AM Rodriguez et al., BBRC ., 315: 255, 2004). These adipose tissues have the advantage of being able to be extracted in large quantities, and are attracting attention as a source of new stem cells to supplement the existing disadvantages. In addition, recent studies have shown that adipose tissue-derived cells have the ability to promote muscle regeneration and neurovascular differentiation through animal model experiments, and have emerged as a new source of stem cells.

Recently, there is a great interest in the identification, isolation and development of such human stem cells. Human stem cells are totipotential or pluripotential precursor cells capable of generating a variety of adult cell lineages. This ability serves as the basis for the differentiation and specialization of cells required for organ and tissue development. Recently, with the success of such stem cell transplantation, new clinical tools have been provided to reconstruct and / or supplement bone marrow after myeloablation due to exposure to disease, toxic chemicals and / or radiation. There is additional evidence to demonstrate that stem cells can be used to repopulate many, but not all, tissues and restore physiological and anatomical function. In addition, the application of stem cells in tissue engineering, gene therapy delivery and cell therapy products is rapidly progressing. Therefore, as the interest in stem cells increases in the art, there is an urgent need for the development and manufacture of stem cells from various tissues.

Accordingly, the present inventors have made efforts to prepare stem cells in various tissues. As a result, when preparing placental stem cells using placental tissues, compared to preparing stem cells using conventional adipose tissue, It was confirmed that the production can be more efficient, and completed the present invention.

An object of the present invention is characterized in that the fine amnion, membrane, basal decidual or placental tissues are recovered after culturing in bFGF (Basic Fibroblast Growth Factor) containing medium, and (a) CD29, CD44, CD73, CD90 and CD105. All show positive immunological properties, and all show negative immunological properties for CD31, CD34, CD45 and HLA-DR; (b) exhibits positive immunological properties for Oct4 and SSEA4; (c) adhered to plastic and grown, exhibiting morphological properties of round-shape or spine-shape, forming spheres in SFM media and allowing long-term maintenance in undifferentiated state; And (d) a method for producing placental stem cells having the ability to differentiate into mesoderm, endoderm and ectoderm derived cells, and obtained placental stem cells.

Another object of the present invention is a method for differentiating from pluripotent placental stem cells to myocytes, neurons, osteoblasts, adipocytes, chondrocytes and pancreatic beta cells and containing the differentiated cells or the placental stem cells To provide a cell therapy for the treatment of osteoarthritis, osteoporosis and diabetes, and a cell therapy for the formation of breast tissue.

In order to achieve the above object, the present invention comprises the step of culturing the recovered amnion, membranes, basal decidual or placental tissue in the collagenase and bFGF-containing medium, and then recovering (a) CD29, CD44, CD73, Both show positive immunological properties for CD90 and CD105 and all show negative immunological properties for CD31, CD34, CD45 and HLA-DR; (b) exhibits positive immunological properties for Oct4 and SSEA4; (c) adhered to plastic and grown, exhibit morphological properties of round- or spine-shape, and form spheres in SFM media to allow long-term maintenance in undifferentiated state; And (d) provides a method for producing placental stem cells and the placenta stem cells obtained thereby having the ability to differentiate into mesoderm, endoderm and ectoderm derived cells.

Also provided is a method of differentiating muscle cells, osteogenic cells, neurons, adipocytes, chondrocytes, or pancreatic beta cells from the obtained placental stem cells.

The present invention also provides a cell therapeutic agent containing the obtained placental stem cells or cells differentiated therefrom as an active ingredient.

Hereinafter, the present invention will be described in detail.

1. Definition of terms

As used herein, the term 'placental stem cell' refers to a cell that is not derived from the inner cell mass of the blastocyst. Stem cells that can be obtained from the placenta include placental stem cells, pluripotent cells, pluripotent cells, and progenitor cells.

As used herein, the term 'pluripotent cell' refers to a cell that has the ability to grow into any subset of about 260 cell types of the mammalian body. Unlike pluripotent cells, pluripotent cells do not have the ability to form all cell types.

As used herein, the term 'progenitor cell' refers to a cell that is intended to differentiate into a specific type of cell or form a specific type of tissue.

As used herein, the term 'stem cells' refers to master cells that can be regenerated without limitation to form specialized cells of tissues and organs. Stem cells are developable pluripotent or pluripotent cells. Stem cells can divide into two daughter cells, or one daughter cell and one derived ('transit') cell, and then proliferate into mature, fully formed cells of the tissue.

As used herein, the term "differentiation" refers to a phenomenon in which structures or functions are specialized while cells divide and proliferate and grow, that is, a cell or tissue of an organism has a shape or function to perform a task given to each. It means to change. Generally, a relatively simple system is divided into two or more qualitatively different sub systems. For example, qualitatively between parts of a living organism that were almost homogeneous in the first place, such as head or torso distinctions between eggs that were initially homogenous in the development, or cells such as muscle cells or neurons. Phosphorus difference, or as a result, is a state divided into subclasses or subclasses that can be distinguished qualitatively.

As used herein, the term 'cell therapeutic agent' is a medicinal product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special chewing from humans, and functions of cells or tissues. Refers to a drug used for therapeutic, diagnostic and prophylactic purposes through a series of actions, such as the proliferation and selection of living autologous, allogeneic, or heterologous cells in vitro or by altering the biological characteristics of the cell in order to restore the . Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.

2. Isolation and Purification of Placental Stem Cells

The placenta is made for the fetus during pregnancy, and has a disc shape of 500g in weight, 15-20cm in diameter, and 2-3cm in thickness. One side of the placenta is in contact with the mother and the other is in contact with the fetus, and the space between the mother's blood contains the nourishment to the fetus. The placenta is composed of three layers of amnion, mesentery and decidual membrane. In addition, the amniotic membrane is a thin transparent membrane surrounding the fetus, which contains amniotic fluid, and the amnion contains fetal stem cells. The decidual membrane is a membrane formed by deforming the epithelial cells of the uterus so that the fertilized egg is implanted in the uterus. The amount of stem cells in the placenta is very abundant, proliferates and can differentiate into other cells.

Decidua or amnion-derived stem cells isolated from human term placenta according to the present invention are classified as adult human stem cells and are not ethically problematic because they use placental tissue. Do not.

Usually, the following methods are used to isolate and purify pluripotent stem cells from placental tissue. The present invention relates to the placenta of mammals, preferably to the placenta of humans, which after treatment from the uterus are treated and cultured to produce pluripotent stem cells, placental stem cells and other biological materials. Placental stem cells are obtained from the placenta, which is removed from the uterus. In a preferred embodiment, the placenta is cultured in the presence of growth factors such as Basic Fibroblast Growth Factor (bFGF) and Epidermal Growth Factor (EGF).

In the present invention, first, the placenta is collected from normal and preterm labor at Guro Hospital, affiliated with Korea University Hospital, according to the guidelines of the Institutional Review Board of Korea University Hospital. Purified.

Amniotic and decidual membranes are separated from human placental tissue samples and washed with PBS, respectively. Finely cut washed amnion and decidual tissue. Finely chopped amniotic and decidual tissues are transferred to 100 mm dishes and subjected to chemical degradation for 1 hour at 37 ° C using DMEM (Dulbecco's Modified Eagle Medium, Gibco) medium with collagenase (1 mg / ml).

The chemically decomposed tissues were filtered through a 100 μm mesh to remove the undecomposed tissues, followed by centrifugation for 1 to 10 minutes at 1200 rpm. The supernatant was suctioned and the pellet remaining on the bottom was washed with PBS and centrifuged for 1-10 minutes at 1200 rpm. The pellet at the bottom is well suspended in single cells, and then cultured in a DMEM medium containing bFGF. At this time, the mesenchymal stem cells are attached to the bottom and the other cells are floating.

These stem cells adhere to and grow on plastics and exhibit morphological properties of round-shape or spindle-shape. After two days, the cells that did not adhere to the bottom of the dish were washed with PBS, and cultured while replacing the medium every two to three days to obtain amnion or decidual-derived multipotent stem cell fluid isolated from the human placenta.

Examining the proliferation rate of the isolated placenta-derived pluripotent stem cells, it can be seen that until the passage number (passage number) up to 12, CPDL gradually increases to have an excellent proliferation rate (Figs. 11 and 12).

Stem cells obtained can form a sphere in the SFM medium (sphere) can be maintained in an undifferentiated state for a long time (Fig. 4). One of the SFM medium usable in the present invention contains 10 μM, 1 x antibiotic antimycotic solution, 1 μg / ml hydrocortisone, 5 μg / ml Insuline, 20 ng / ml EGF, 40 ng / ml FGF, B27, β-mercaptoethanol MEBM (Mammary Epithelial Basal Medium) medium is mentioned.

The number and type of proliferated cells can be determined by flow cytometry, cell sorting, immunocytochemistry (eg staining with tissue specific or cell-label specific antibodies), fluorescence activated cell sorting (FACS), Morphology using standard cell detection techniques such as magnetic activated cell sorting (MACS) and measuring changes in the cell's surface label, or by using optical or confocal microscopy Or can be easily monitored by measuring changes in gene expression using techniques well known in the art such as PCR and gene expression profiling.

In a preferred embodiment, the cells cultured in the placenta are subjected to techniques known in the art, such as density gradient centrifugation, magnetic cell separation, flow cytometry, or other cell separation methods, or to methods known in the art. Are classified using.

For example, the FACS method using a flow cytometer with a sorting function ( Int. Immunol. , 10 (3): 275) is a method for obtaining a pluripotent stem cell expressing the target surface antigen from the obtained placental stem cell fluid . , 1998), a method using magnetic beads, and a panning method using an antibody that specifically recognizes pluripotent stem cells ( J. Immunol. , 141 (8): 2797, 1998). In addition, as a method for obtaining pluripotent stem cells from a large amount of culture solution or the like, a method in which an antibody that is expressed on the surface of a cell and specifically recognizes a molecule (hereinafter referred to as a surface antigen) is used alone or in combination and used as a column. There is this.

Examples of the flow cytometer sorting method include a water charge method, a cell capture method, and the like. In one embodiment, the cell surface label-specific antibody or ligand is labeled with a clear fluorescent label. The cells are processed through a cell sorter to separate the cells based on their ability to bind to the antibodies used. FACS-sorted particles can be deposited directly into individual wells of 96-well or 384-well plates to facilitate separation and cloning.

By any method, the antibody that specifically recognizes the surface antigen of the cell is labeled with fluorescence, the fluorescence of the conjugate of the labeled antibody and antigen is measured, and the fluorescence intensity is converted into an electrical signal to quantify the antigen expression level of the cell. Can be. In addition, by combining the kinds of fluorescent materials to be used, it is possible to separate cells expressing a plurality of surface antigens. Fluorescent materials usable here include FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (allo-phycocyanin), TR (TexasRed), Cy3, CyChrome, Red613, Red670, TRI-Color, QuantumRed and the like.

As a FACS method using a flow cytometer, which is one method that can be used in the present invention, the stem cell solution obtained above is collected, the cells are separated by centrifugation, etc., and then stained directly with an antibody. After culturing and propagating in a medium, the method of staining an antibody can be used. To stain the cells, firstly, the primary antibody recognizing the surface antigen and the desired cell sample are mixed and incubated for 30 minutes to 1 hour on ice. If the primary antibody is labeled with fluorescence, it is separated by a flow cytometer after washing. In the case where the primary antibody is not fluorescently labeled, after washing, the fluorescently labeled secondary antibody having a binding activity to the primary antibody and the cells reacted with the primary antibody are mixed and incubated in ice water for 30 minutes to 1 hour. After washing, the cells stained with the primary antibody and the secondary antibody are separated by flow cytometry.

3. Characteristics of Placental Stem Cells Obtained from Placenta

Stem cells isolated from the placenta are homogeneous and sterile. In addition, stem cells are readily obtained in a form suitable for human administration (ie, pharmaceutical grade).

After prolonged incubation, the cells were treated with surface CD series antigen markers such as CD29 (mononuclear cell marker), CD31 (endothelial cell and stem cell marker), CD34, CD44 (hematopoietic cell marker), CD90 (mononuclear stem cell marker), CD73 (T-cell and B-cell marker) Characterized with CD105 (endothelial cell marker), CD45 (hematopoietic cell marker), and can be applied to FACS analysis.

Preferred placental stem cells obtained by the method of the present invention can be characterized by the presence of the following cell surface markers: all show positive immunological properties for CD29, CD44, CD54, CD73, CD90 and CD105, , CD34, CD45 and HLA-DR all show negative immunological properties. Such cell surface labels are measured collectively, for example, by flow cytometry, washed according to methods well known in the art, and stained with anti-cell surface labeled antibodies.

In addition, placental stem cells of the present invention can be identified using a marker of Oct4 or SSEA4, which can be said to be an undifferentiated cell marker (FIG. 9). Oct4 is well known as an undifferentiated state marker in stem cells, and in the art, Korean Patent Application No. 10-2004-0105716 "monoclonal antibody specific for human embryonic stem cells", Korean Patent Application No. 10-2004-0096780 "Double-stranded RNA for inhibiting Oct4 gene expression in maintaining undifferentiated state of mammalian embryos and stem cells," Korean Patent Application No. 10-2006-0092128 And the method for the preparation thereof, etc., in order to prove that the stem cells are in an undifferentiated state, it is common to perform an Oct4 expression test. In addition, it is also known to those skilled in the art that SSEA4 (Stage Specific Embryonic Antigen 4) is present on the surface of human embryonic stem cells.

Expression of Oct4 and SSEA4 uses RT-PCR (Reverase Transcriptase-Polymerase Chain Reaction). The method of RT-PCR is a technique known in the art. RT-PCR is a technique of synthesizing a corresponding cDNA using a template of RNA of a specific site, and then PCR amplification using the same, the experimental process (1) using reverse transcriptase The process consists of preparing cDNA from RNA and (2) amplifying a specific region using cDNA. (2) process is the same as amplifying a specific gene region from genomic DNA. This method is not only simpler than the RNA analysis that was possible through methods such as Northern blot hybridization, but also helps in the study of mRNA sequencing and transcription, since the gene sequence can be determined. This is the technology.

Placental stem cells of the present invention show a positive response to the expression of Oct4 and SSEA4 (FIG. 9).

4. Differentiation of Placental Stem Cells

Placental stem cells obtained by the method of the present invention have the ability to differentiate into mesoderm, endoderm and ectoderm derived cells. It can be induced to differentiate along specific cell lineages, including adipocyte differentiation, chondrocyte differentiation, osteoblast differentiation, hematopoietic cell differentiation, myocyte differentiation, vascular cell differentiation, neuronal differentiation, hepatocyte differentiation. In certain embodiments, placental stem cells obtained according to the methods of the present invention are induced to differentiate for use in transplantation and ex vivo treatment protocols. In certain embodiments, placental stem cells obtained according to the methods of the invention are induced to differentiate into specific cell types and are genetically designed to provide a healing gene product.

In certain embodiments, the placental stem cells obtained by the methods of the invention are incubated with an ex vivo compound that is induced to differentiate and then the differentiated cells are transplanted directly into the subject. Thus, the present invention includes a method of differentiating human placental stem cells using standard culture media. Thus, pluripotent placental stem cells according to the present invention include a method of differentiating myocytes, neurons, astrocytes, osteoblasts, chondrocytes, adipocytes and insulin secreting pancreatic beta cells.

Determination of the differentiation of stem cells into specific cell types can be performed by methods well known in the art, for example, (1) SKBM medium after placenta stem cells are pretreated with azacytidine for one day. (cambrex, co.) can induce differentiation into myocytes, (2) placental stem cells are pre-cultured in DMEM medium containing BME (beta-mercaptoethanol) and FBS (Fetal Bovine Serum), and the preculture Can be treated with DMSO (dimethyl sulfoxide) and BHA (Butylated hydroxyanisole) to induce neuronal differentiation, (3) placental stem cells can be mixed with TCP (Tricalcium phosphate) and isotransplanted to differentiate into osteoblasts, (4) Placental stem cells can be differentiated into adipocytes by culturing in α-MEM medium containing dexamethasone, indomethacin, insulin and IBMX (3-isobutyl-1-methylxanthine).

The differentiation may also be performed using techniques such as flow cytometry or immunocytochemistry to measure cell surface labeling (e.g., staining cells with tissue-specific or cell-labeling specific antibodies) and morphological changes, Or by examining the morphology of the cells using confocal microscopy, or by measuring changes in gene expression using techniques well known in the art such as PCR and gene-expression profiles.

6. Use of placental stem cells and their differentiated cells

Placental stem cells of the present invention can be used in various types of therapeutic protocols in which tissues or organs of the body are enriched, treated or replaced by engraftment, transplantation or infusion of the desired cell population, for example stem cells or derived cell populations. have. The placental stem cells of the present invention can replace or enhance existing tissue, resulting in new or changed tissue, or bind to biological tissue or structure. In addition, embryonic stem cells may typically be replaced with placental stem cells of the invention in therapeutic protocols in which embryonic stem cells are used. For example, Alzheimer's disease containing placental stem cells or differentiated cells of the present invention, neurological diseases such as Parkinson's disease, progressive muscular dystrophy, muscular diseases such as Lou Gehrig's disease, osteoarthritis, osteoporosis bone diseases and diabetes treatment cells It can be used for therapeutic agents and cell therapies for forming breast tissue.

In a preferred embodiment of the invention, placental stem cells and other stem cells from the placenta can be used for autologous and allogeneic hematopoietic transplantation, including suitable and incompatible HLA type hematopoietic transplantation. For example, the placental stem cells of the present invention may be used in therapeutic transplantation protocols such as liver, pancreas, kidney, lung, nervous system, muscular system, bone, bone marrow, thymus, spleen, submucosal tissue, gonads or hair stem cells or It can be used to enhance or replace derived cells.

Placental stem cells are typically used in place of certain classes of progenitor cells (eg, chondrocytes, stem cells, hematopoietic cells, pancreatic parenchymal cells, neural stem cells, and muscle derived cells, etc.) in the therapeutic or research protocol in which the derived cells are used. Can be.

Placental stem cells of the present invention can be used to strengthen, treat or replace cartilage, tendons or ligaments. For example, in certain embodiments, prostheses (eg, buttock prostheses) are coated with replacement cartilage structures grown from placental stem cells of the invention. In other embodiments, the joint (eg, knee) is reconstituted with cartilage structures grown from placental stem cells. In addition, cartilage structures can be used primarily for reconstructive surgery of different types of joints (eg, the protocol in Resnick, D. et al, Diagnosis of Bone and Joint Disorders, 2d, 1988).

Placental stem cells of the present invention can be used to repair damage to tissues and organs resulting from disease. In such embodiments, the placenta stem cells may be administered to the patient to regenerate or repair the damaged tissues or organs as a result of the disease (eg, to improve the immune system following chemotherapy or radiation, and cardiac tissue after myocardial infarction). To recover).

The placental stem cells of the present invention can also be formulated as injectables (eg, WO 96/39101, incorporated herein by reference). In other embodiments, the cells and tissues of the present invention are described in U.S. Patents 5,709,854; 5,516,532; 5,516,532; It may be formulated using polymerizable or crosslinkable hydrogels as described in US Pat. No. 5,654,381, all of which are incorporated herein by reference.

Through the following examples will be described the present invention in more detail. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation and Separation of Placental Tissues

Placenta was collected from normal and preterm labor at Guro Hospital, Korea University, according to the guidelines of the Institutional Review Board of Korea University. Placental tissues were transferred to the lab in physiological saline containing antibiotics.

Placental tissues that have been transferred to the laboratory are washed with PBS to remove blood cells and debris from various tissues, or tissues are removed using hemolysis buffer to remove blood cells or amnion and membranes that make up the placenta. (chorion), decidual (decidua) and placenta bad tissues were carefully separated using a facet, respectively.

Example 2: Isolation and Culture of Placental Stem Cells

Each tissue was separated into 100mm dishes and chopped into 1 ~ 2mm size using a sterile mesh. Thereafter, it was placed in the culture medium containing collagenase and reacted for at least 1 hour up to 4 hours in an incubator at 37 ° C., and the collagenase-treated tissue was filtered using 100 wire fabrics. The cells thus isolated were incubated in a 100 mm dish in DMEM medium containing bFGF under 37 ° C. and 5% CO ₂ .

Comparative Example 1: Comparison of Stem Cell Culture Efficiency of Placental and Adipose Tissues

When stem cells were separated and cultured from 5 g of adipose tissue, more than 10 million cells were obtained after two passages, but about 3 g of placental tissues were able to obtain more than 10 million cells after the first passage. Through this, it was found that culturing stem cells using placental tissue was much more efficient than culturing stem cells using conventional adipose tissue.

Example 3 Investigation of Proliferation Rate of Placental Tissue-Derived Stem Cells

The proliferation rate of the stem cells obtained according to the proliferation method of the isolated human placental tissue-derived pluripotent stem cells was investigated. Placental stem cells obtained from placental tissue samples of different human subjects were obtained through the isolation method as in Examples 1 to 3, and then seeded by 2 × 10 ⁵ by 75-flask.

Cumulative population doubling level (CPDL) is an index indicating the proliferation rate of cells.

CPDL = ln (Nf / Ni) / ln2

(Ni = initial seeded cell number, Nf = final cell number)

It can be expressed as

CPDL values of basal decidual-derived stem cells and amnion-derived stem cells were observed according to passage numbers, and when the passage number was 12, the CPDL values reached approximately 30 (FIGS. 11 and 12). These CPDL values are similar to those of human adipose tissue derived stem cells (Lin et al., Stem cells and development, 14:92, 2005; Zuk et al., Tissue eng., 7: 211, 2001). From the adult stem cells according to the present invention was found that the proliferation rate is very excellent.

Example 4: Immunological Characteristics of Placental Pluripotent Stem Cells

Placental tissue-derived pluripotent stem cells obtained in Example 3 were washed with PBS, trypsinized, and the cells were harvested and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, washed with 5% FBS and PBS mixed, and centrifuged at 1000 rpm for 5 minutes. After discarding the supernatant, the cells were suspended in PBS and 1 × 10 ⁵ cells were dispensed as many as the number of samples. In each well, an antibody (R-phycoerythrin-conjugated mouse anti-human monoclonal antibody) was added and incubated on ice for 40 minutes. After incubation it was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, washed with PBS and centrifuged at 1000 rpm for 5 minutes. Once again, the supernatant was removed and washed with PBS and centrifuged for 5 minutes at 1000 rpm. After the supernatant was removed, 1% paraformaldehyde was added and singled, and analyzed using a flow cytometer. As a result, as shown in Table 1 below, placental tissue-derived placental stem cells of the present invention showed positive immunological properties for CD29, CD44, CD73, CD90 and CD105, and CD31, CD34, CD45 and HLA-. It can be seen that all of the DRs showed negative immunological characteristics.

Surface antigen analysis of placental stem cells Antigen AD-MSCs CD29 + CD31 - CD44 + CD90 + CD105 + CD34 - CD45 - CD73 + CD34 - HLA-DR -

Example 5 Analysis of Oct4 and SSEA Expression in Placental Pluripotent Stem Cells

Placental stem cells obtained in Example 3 were washed three times with PBS, and fixed for 30 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, it is permeabilized with PBS containing 0.1% Triton-X100 for 10 minutes. After washing three times with PBS, the reaction was treated with Blocking buffer (5% goat serum) for 1 hour at 4 ℃, overnight to the blocking buffer containing the primary antibody. Washed three times with PBS, and reacted with a secondary antibody in the dark for 1 hour. After washing three times with PBS, it was mounted. As a result, as shown in Figure 9, the differentiating stem cells according to the present invention showed a positive response to Octa and SSEA markers of human embryonic stem cells (Fig. 10).

In addition, the expression of Oct4 was confirmed using RT-PCR. RT-reaction was performed at 37 ° C. for 50 minutes, 70 ° C. for 10 minutes, and PCR-reaction was performed at 95 ° C. for 5 minutes, and at 95 ° C. for 30 seconds / 40 cycles were performed at 58 ° C. for 40 seconds / 72 ° C. for 1 minute, followed by 10 minutes at 72 ° C. As a result, as shown in Figure 9 it can be confirmed that the basal decidual-derived stem cells and amnion-derived stem cells are expressed at 800bp.

Example 6: Differentiation of Placental Pluripotent Stem Cells into Myocytes

Placental tissue-derived pluripotent stem cells obtained in Example 3 were dispensed into 10 ng / ml fibronectin-coated flasks and pretreated for 24 hours using 10 μM 5′-azacytidine. do. After pretreatment, the cells were incubated for 10 days using SKBM medium (Cambrex, Co.), followed by immunostaining.

As a result, human placental tissue-derived pluripotent stem cells according to the present invention showed a positive response to myosin, a specific antigen of muscle cells. From these results, it was confirmed that the human placental tissue-derived pluripotent stem cells according to the present invention were differentiated into muscle cells.

Example 7: Differentiation of Placental Tissue Pluripotent Stem Cells into Neurons

Placental tissue-derived pluripotent stem cells obtained in Example 3 were preincubated for 1 day using DMEM medium containing 1 mM BME and 10% FBS. Differentiation into neurons was induced in neuronal differentiation induction medium containing 1% DMSO and 100 μM BHA after preculture. Nine days after the addition of neuronal differentiation induction medium, immunostaining was performed.

As a result, human placental tissue-derived pluripotent stem cells according to the present invention showed a positive response to GFAP (glial fibrillary acidic protein), which is a specific antigen of neural astrocytes. From these results, it was confirmed that the human placental tissue-derived pluripotent stem cells according to the present invention were differentiated into neurons (FIG. 6).

Example 8 Differentiation of Placental Pluripotent Stem Cells into Osteoblastic Cells

Placental tissue-derived placental stem cells obtained in Example 3 were subjected to bone formation induction medium (0.1 μmol / L dexamethasone, 0.05 mmol / L ascorbic acid-2-phosphate, and 10 mmol / L beta-glycophsphate, 5-30% human serum). Or dilution in plasma) to count the cells, and then cultured in flasks (5% CO 2, 37, replacing the medium once every 3-4 days) to induce differentiation into osteogenic cells. At 14 days after the start of the culture, it was confirmed that the placental-derived pluripotent stem cells were differentiated into osteogenic cells using Alizalin red S staining (FIG. 7).

Example 9 Differentiation of Placental Tissue Pluripotent Stem Cells into Adipocytes

Placental tissue-derived pluripotent stem cells obtained in Example 3 were prepared in α-MEM medium containing 5% FBS, 1 μM Dexamethasone, 200 μM Indomethacin, 10 μg / μL Insulin, and 0.5 mM IBMX (3-isobutyl-1-methylxanthine). Cultured for 2 weeks to induce differentiation of pluripotent stem cells into adipocytes, and analyzed using Oil red O staining method. As a result, it was confirmed that the human placental tissue-derived pluripotent stem cells according to the present invention were differentiated into adipocytes (FIG. 8).

As described above in detail, the method for producing placental stem cells using placental tissue according to the present invention can be produced more efficiently, compared to the production of stem cells from other existing tissues, and the pluripotent stem according to the present invention. The cells have the ability to differentiate into myocytes, vascular endothelial cells, osteoblasts, neurons, astrocytes, adipocytes, chondrocytes, osteoblasts, or insulin-secreting pancreatic beta cells, and thus, cirrhosis, osteoporosis, osteoarthritis, It is effective in the treatment of neurological diseases, diabetes and the like, and useful for forming breast tissue.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (21)

A method for the preparation of adult stem cells having the following characteristics, characterized in that the amnion, membranes, basal decidual membranes or placental tissues of human placental tissues are cultured in bFGF-containing medium and then recovered. (a) all showed positive immunological properties for CD29, CD44, CD54, CD73, CD90 and CD105 and all negative immunological properties for CD31, CD34, CD45 and HLA-DR; (b) exhibits positive immunological properties for Oct4 and SSEA4; (c) adheres to plastic and grows, exhibits morphological characteristics of round-shape or spine-shape, and forms spheres in SFM medium to remain undifferentiated; And (d) Has the ability to differentiate into mesoderm or ectoderm derived cells. Placental stem cells obtained by the method of claim 1 having the following characteristics: (a) all positive immunological properties for CD29, CD44, CD73, CD90 and CD105, and all negative immunological properties for CD31, CD34, CD45 and HLA-DR; (b) exhibits positive immunological properties for Oct4 and SSEA4; (c) adheres to plastic and grows, exhibits morphological characteristics of round-shape or spine-shape, and forms spheres in SFM medium to remain undifferentiated; And (d) Has the ability to differentiate into mesoderm or ectoderm derived cells. The placental stem cell according to claim 2, wherein the mesoderm-derived cells are osteoblasts, chondrocytes, myocytes or adipocytes, and the ectoderm-derived cells are neurons. The method of differentiating placental stem cells into muscle cells, characterized in that the placental stem cells of claim 2 are pretreated with azacytidine for one day and then cultured in SKBM medium. A cell therapeutic agent for the treatment of muscle diseases containing myocytes differentiated by the method of claim 4 as an active ingredient. A method of differentiating placental stem cells into neurons, comprising the following steps: (a) pre-culture of placental stem cells of claim 2 in DMEM medium containing BME and FBS; And (b) treating the preculture with DMSO and BHA to induce neuronal differentiation. A cell therapeutic agent for the treatment of neurological diseases, comprising as an active ingredient neurons differentiated by the method of claim 6. Placental stem cells of claim 2 is mixed with TCP (Tricalcium phosphate) is characterized in that the transplantation, placental stem cells differentiation into osteoblasts. Cell therapy for osteoporosis treatment containing osteogenic cells differentiated by the method of claim 8 as an active ingredient. The method of differentiating placental stem cells into adipocytes, wherein the placental stem cells of claim 2 are cultured in an α-MEM medium containing dexamethasone, indomethacin, insulin and IBMX. A cell therapeutic agent for forming breast tissue, comprising the adipocytes differentiated by the method of claim 10 as an active ingredient. delete delete delete delete A cell therapeutic agent for the treatment of muscle diseases containing placental stem cells of claim 2, which has the property of differentiating into muscle cells. Cell therapy agent for the treatment of neurological diseases, comprising placental stem cells of claim 2 having the characteristics of differentiating into nerve cells as an active ingredient. Cell therapy for osteoarthritis, comprising placental stem cells of claim 2 having the characteristics of differentiating into chondrocytes as an active ingredient. A cell therapeutic agent for treating osteoporosis, comprising the placental stem cell of claim 2 as an active ingredient having the property of differentiating into osteogenic cells. A cell therapeutic agent for forming breast tissue, comprising the placental stem cell of claim 2, which has the property of differentiating into adipocytes, as an active ingredient. delete

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