KR20080068394A - ADULT STEM CELLS HAVING AN ABILITY OF DIFFERENTIATION INTO PANCREAS Beta; CELLS - Google Patents

Abstract

An adult stem cell derived from a human tissue is provided to be differentiated into beta cells, thereby being useful for cell therapy of diabetic diseases. A method for differentiating adult stem cells into pancreas beta cells comprises a step of culturing adult stem cells derived from a human tissue in a medium not including nestin positive cells, wherein the adult stem cell derived from the human tissue is prepared by culturing monocytes isolated from the human tissue in a culture medium including bFGF, penicillin, streptomycin, glutamine and FBS and then recovering the culture material and the adult stem cell is characterized in that it has Oct4 and SSEA4 expression capability, shows positive immunological characteristics on CD29, CD31, CD44, and CD45 and negative immunological characteristics on CD4, CD20, CD34, and CD51, is attached to a plastic to grow, shows morphological characteristics of round shape or spindle shape, has the ability of being differentiated into cells derived from endoderm, mesoderm and ectoderm, and has a characteristic of being differentiated into the pancreas beta cells. A cell therapeutic agent for treating diabetes comprises the pancreas beta cell as an effective ingredient. Further, the adult stem cell derived from the human tissue is a adult stem cell derived from a cord blood.

Description

Adult Stem Cells Having an Ability of Differentiation into Pancreas β Cells}

1 a is a colony photograph formed from a collection of human umbilical cord blood-derived adult stem cells (which share specific properties of embryonic stem cells), and Oct4 (b), which is an embryonic stem cell specific expression protein in human umbilical cord blood-derived adult stem cells, It is a photograph confirming the expression of SSEA4 (c).

Figure 2 is an immunostained picture for each factor [a and d: C-peptide (red); b and e: insulin (green); c and f: pancreatic beta-cell-like structure]

Figure 3 shows the Western blot analysis of C-peptide (a) and Oct4 (b).

The present invention provides a method for differentiating adult stem cells into pancreatic beta cells (insulin producing cells), comprising culturing adult stem cells derived from human tissues including cord blood in a medium without nestin positive cells. It relates to the pancreatic beta-like cells obtained.

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 (Jiang, Y. et. al ., Nature , 418: 41, 2002), and have since been identified in several other adult tissues (Verfaillie, CM et. al , 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, muscles and brain (Toma, JG et al ., Nat . Cell Biol ., 3: 778, 2001; Sampaolesi, M. et al., Science , 301: 487, 2003; Jiang, Y. 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. In other words, it is very difficult to separate stem cells and preserve them in vitro.

Umbilical cord blood has recently been reported to have a large amount of stem cells, and research is being actively conducted. Since cord blood is known to be a rich source of hematopoietic stem cells, many clinical trials have been attempted to treat blood-related diseases through cord blood transplantation. Banks are also active in Korea. Umbilical cord blood, unlike the bone marrow, can be obtained through a simple procedure in the umbilical cord (Umbilical cord) that is discarded during the delivery process, and contains a large number of hematopoietic stem cells and stem cells compared to the amount.

Compared with peripheral blood transplantation, human cord blood transplantation is a much more promising method for treating pediatric and adult patients because of rapid availability, low risk of infectious disease transmission, and graft-versus-host disease. Because of the less risk. Umbilical cord blood transplantation has been well studied so far and is used for the treatment of diseases related to hematopoietic dysfunction.

McGuckin et al. Isolated SSEA-3 +, SSEA-4 + and Oct4 + embryonic stem cell-like cells from non-hematopoietic cell populations of cord blood (CD45-) [McGuckin, CP et. al ., Cell Proliferation 38, 245-255, 2005]. Recently, Yong Zhao et al. Have described human cord blood that exhibits embryonic stem cell-like properties, including expression of embryonic stem cell specific molecular markers, along with SSEA-3 and SSEA-4 as well as the transcription factors Oct4 and Nanog. A cluster of stem cells for was obtained [Zhao, Y. et al ., Experimental Cell Research , 312: 2454-2464, 2006]. Polesskaya et al. Suggested that mobilization of intrinsic CD45 positive cells is an important factor in regeneration after injury once they are activated by the Wnt pathway [Polesskaya, A. et al. , Cell , 113: 841-852, 2003]. Yeh SP and others also reported that CD45 is expressed by bone marrow-derived mesenchymal stem cells at various levels [Yeh, SP, et. al ., Leukemia, 19: 1505-1507, 2005]. All these evidences suggest that CD45 is not only a marker of blood-lineage cells but also is expressed in stem cells.

Differentiation from embryonic stem cells to insulin producing cells as well as other types of stem cells in vitro is well studied. Despite the majority of protocols that induce beta cell differentiation following the selection of nestin expressing cells, Przemyslaw et al. Developed a new protocol without nestin positive cell selection [Przemyslaw B. et. al . , Int. J. Dev . Biol ., 48: 1095-1104, 2004]. Since the cell colonies of the present invention exhibit certain embryonic stem cell (ESC) properties, similar protocols have been applied to induce them into beta cell differentiation. No insulin or C-peptide was detected without high glucose conditions of activity. Yong Zhao et al. Demonstrated that human umbilical cord blood-derived stem cells can produce therapeutic glycemic effects in STZ mouse models with diabetes [Zhao Y et et al. al . , Cell Research , 312: 2454-2464, 2006]. In vitro differentiation into insulin producing pancreatic cell-like structures provides new evidence to demonstrate stem cell properties of cord blood derived cells.

Diabetes mellitus (DM), on the other hand, is a disease that causes high morbidity and mortality in many countries and is associated with type 1 diabetes and reduced insulin sensitivity due to absolute insulin deficiency caused by the destruction of insulin-branched pancreatic cells. Type 2 diabetes due to relative insulin deficiency. Recently, cell replacement therapy for diabetes, especially Type 1 diabetes, has received much attention. However, transplantation of pancreatic beta cells can free many patients from exogenous insulin infusion, but there are not enough organ donors and patients have to risk anti-rejection therapies [ Lewis EC et al . , Cytokine , 34: 106-113, 2006].

Today, much effort is being made to find alternative means of treating diabetes through stem cell therapy. Stem cells are a group of cells that have self-renewal ability and are capable of differentiating into many kinds of lineage cells. Many reports have been made regarding the differentiation of many types of stem cells into insulin-producing pancreatic beta cells, including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells and pancreatic stem cells. Most of these methods are carried out via a multi-step protocol based on nestin selection. Stem cells are transplanted into patients before or after differentiation into insulin producing cells to correct hyperglycemic conditions.

Embryonic stem cells are thought to be able to differentiate into any kind of lineage cells, but ethical limitations and limited sources have hampered research on fetal stem cells. In addition, although insulin producing cells can be produced by embryonic stem cells, cases of malformation have been reported after transplantation into animals. Adult stem cells such as bone marrow mesenchymal stem cells and pancreatic stem cells have high immunity as their inherent limitations. Fetal stem cells, particularly cord blood stem cells, have advantages as a means of stem cell therapy primarily in the clinic because of their low immunity and relatively easy source acquisition.

Only a few papers report on the clinical application of human umbilical cord blood (UCB) stem cells. However, in our previous study, UCB stem cells were applied in patients suffering from spinal cord injury and Burger's Disease. Shuro Yoshida et al. Have successfully demonstrated that UCB-derived stem cells can produce insulin-producing cells in NOD / SCID / ₂ m ^null mice with fewer mature T and B cells and showing extremely low activity of natural killer (NK) cells. Yoshida S. et al . , Stem Cells, 23; 1409-1416, 2005]. Coupled with the global shortage of transplant-prepared somatic cells, the success of therapy based on islet cell transplantation for type I diabetes has been a stimulus to efforts to develop new sources of islet cell replacement tissue. In several studies, fetal stem cells (ESCs) have been successfully induced into pancreatic islet-like structures that produce insulin. However, there are ethical and technical concerns with ESC's source.

Tai et al. Demonstrated that Oct4 genes are expressed in a number of human adult stem cells, including pancreatic stem cells [Tai, MH et. al ., Pancreas , 26: e18-e26, 2003]. Recently there have been several reports that stem cells derived from human umbilical cord blood share some characteristics of embryonic stem cells [McGuckin C. P et. al , Cell Proliferation, 38; 245-255, 2005; Zhao Y et al ., Experiment Cell Research , 312: 2454-2464, 2006]. In addition, a method of differentiating adult stem cells into pancreatic beta cells is known using embryonic stem cells (KR10-2004-0068197), or using adult stem cells but using nestin-positive cells (US 20050054102). In addition, there have been reports of methods for making insulin-producing cells from human umbilical cord blood-derived cells using both cell fusion, fusion-dependent and fusion-independent mechanisms [Yoshide S. at al . , Stem Cells , 23: 1409-1416, 2005] The present inventors have tried to find a way to differentiate adult stem cells into pancreatic beta cells in an easier way, and to this day whether embryonic stem cells can be induced into insulin producing cell structure. Since much research has been conducted, the inventors have questioned whether such a process can be applied to stem cells derived from cord blood.

The present inventors thus conceived an unselected protocol of nestin-positive cells by obtaining an embryonic stem cell-like human umbilical cord blood-derived stem cell and then altering a protocol reported to induce embryonic stem cells into pancreatic beta cells, Using this as a medium, it was confirmed that human tissue-derived stem cells can be differentiated into insulin-producing cell structures, thereby completing the present invention.

It is a main object of the present invention to provide a method for differentiating adult stem cells derived from human tissue into insulin-secreting pancreatic beta-like cells and to provide insulin-secreting pancreatic beta-like cells obtained by the method.

Another object of the present invention is to prepare a human tissue-derived adult stem cells comprising the step of culturing the human tissue-derived monocytes in a medium containing bFGF, penicillin, streptomycin, glutamine and FBS and obtained by the method To provide adult stem cells.

It is another object of the present invention to provide a cell therapy agent for treating diabetes containing the adult stem cells derived from human tissues or pancreatic beta-like cells secreting insulin as an active ingredient.

In order to achieve the above object, the present invention is a method for differentiating adult stem cells into pancreatic beta-like cells by culturing adult stem cells derived from human tissues in a medium without nestin positive cells and pancreatic beta obtained by the method. Provide like cells. The pancreatic beta-like cells are characterized in that insulin and C-peptide are expressed but Oct4 is not expressed.

The present invention is also characterized in that the mononuclear cells isolated from human tissues are cultured in a medium containing bFGF, penicillin, streptomycin, glutamine and FBS, and then recovered, (a) having the ability to express Oct4 and SSEA4; (b) all show positive immunological properties for CD29, CD31, CD44, and CD45, and all show negative immunological properties for CD4, CD20, CD34, and CD51; (c) attached to and grown on plastics, exhibiting morphological properties of round-shape or spindle-shape; (d) has the ability to differentiate into mesoderm, endoderm and ectoderm derived cells; And (e) provides a method for producing adult stem cells derived from human tissue exhibiting the characteristics of differentiating into insulin-secreting pancreatic beta-like cells.

The present invention is also obtained by the above method, and (a) has the ability to express Oct4 and SSEA4; (b) all show positive immunological properties for CD29, CD31, CD44, and CD45, and all negative immunological properties for CD4, CD20, CD34, and CD51; (c) attached to and grown on plastics, exhibiting morphological properties of round-shape or spindle-shape; (d) has the ability to differentiate into mesoderm, endoderm and ectoderm derived cells; And (e) human tissue derived adult stem cells having the property of differentiating into insulin secreting pancreatic beta-like cells.

The present invention also provides a cell therapy agent for treating diabetes containing human tissue-derived adult stem cells or differentiated pancreatic beta-like cells having differentiation ability into the pancreatic beta-like cells as an active ingredient.

In particular, the human tissue-derived adult stem cells of the present invention can be selected from human tissue-derived stem cells selected from the group consisting of breast, bone marrow, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, and uterus. Preferably, adult stem cells derived from cord blood may be selected.

1. Definition of terms

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.

The term 'adult stem cells' used in the present invention refers to stem cells that appear during the development or development of each organ of the embryo or adult stage. Adult stem cells are generally limited to cells that make up a specific tissue. These adult stem cells remain in most organs after adulthood and play a role in supplementing the loss of normal or pathological cells.

As used herein, the term 'differentiation' refers to a phenomenon in which structures or functions are specialized during the division and growth of cells, that is, in the form or function of a cell or tissue of an organism to perform a given task. 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 the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or heterologous cells in vitro or by altering the biological characteristics of the cells in order to restore them. . 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 Umbilical Cord Blood-derived Stem Cells

Umbilical cord blood used as an aspect in the present invention is blood collected from the umbilical vein connecting the placenta and the fetus in humans, and is collected before delivery of the placenta. Umbilical cord blood-monocytes (MNCs) were prepared from heparinized umbilical cord blood from single delivery by ficoll-hypaque density gradient centrifugation according to standard assays (T.Tondreau et al. , Mesenchymal Stem Cells Derived from CD133-Positive Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression, and Plasticity Stem Cells 23: 1105, 2005).

Specifically, the cord blood collected after delivery before separation of the placenta is diluted with phosphate buffer (PBS) and diluted, and then superimposed on the diluted cord blood and the same amount of picol-high pack solution. At this time, it is preferable that the Ficoll-Hippack solution is brought to room temperature before use, and that the volume of diluted cord blood does not exceed three times the volume of the Ficol-Hippack solution. It is centrifuged to separate the erythrocyte layer, monocyte layer and serum layer. Pasteur piptte and the like to transfer the mononuclear cell layer only to a new tube, and washed with PBS, etc. to remove the Ficoll-Hippack solution or contaminated platelets incorporated when collecting the mononuclear cell layer.

The medium used for culturing the isolated cells in the present invention is a medium for animal cell culture as a medium for mammalian cell therapy containing inorganic salts, amino acids, vitamins and / or cofactors as a medium for animal cell culture. Medium commonly used for cell culture can be used. Commercially available medium for culturing animal cells includes DMEM (Dulbecco's Modified Eagle Medium).

In the present invention, the DMEM medium is cultured by adding bFGF (basic fibroblast growth factor) [R & D Sytem Minneapolis, MN], penicillin, streptomycin, glutamine (Gibco, grand island, NY) and 20% FBS. These stem cells adhere to and grow on plastics and exhibit morphological properties of round-shape or spindle-shape.

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

For example, FACS method using a flow cytometer with a sorting function ( Int. Immunol. , 10 (3): 275, 1998) is a method for obtaining a pluripotent stem cell expressing the surface antigen of interest from the stem cell solution obtained . ), 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 for 30 minutes to 1 hour in ice water. After washing, the cells stained with the primary antibody and the secondary antibody are separated by flow cytometry.

3. Characteristics of Umbilical Cord Stem-Derived Adult Stem Cells

After prolonged incubation, the cells were treated with surface CD series antigen markers such as CD4 (T cell marker), CD20 (B cell marker), CD29 (mononuclear cell marker), CD31 (endothelial cell and stem cell marker), and CD34 (hematopoietic stem). Characterization of cell markers (CTM), hematopoietic cell markers (CD44), hematolineage cell markers (CD45), monouclear stem cell markers (CD90), endothelial cell markers (CD105), and hematopoietic stem cell markers (CD133) for FACS analysis Applicable

Preferred umbilical cord blood-derived 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, CD31, CD44, and CD45, CD4, CD20, Both CD34, and CD51 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, the umbilical cord blood-derived 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. 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", Korean Patent Application No. 10-2006-0092128 "Multiple stem cells derived from human cord blood with increased proliferative capacity by osteoclast-based nitric-like structures As shown in "Cells and Manufacturing Methods", etc., it is common to perform an Oct4 expression test as an experiment for proving that they are stem cells in undifferentiated state. 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 It 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.

Adult stem cells isolated by the present inventors also showed positive response to Oct4 and SSEA-4 (FIG. 1). In other words, they are adult stem cells that possess the characteristics of embryonic stem cells.

In short, the present invention, in a preferred embodiment, the mononuclear cells isolated from the cord blood-derived blood is cultured in a medium containing bFGF, penicillin, streptomycin, glutamine and FBS and then recovered, (a) has the ability to express Oct4 and SSEA4 ; (b) all show positive immunological properties for CD29, CD31, CD44, and CD45, and all show negative immunological properties for CD4, CD20, CD34, and CD51; (c) attached to and grown on plastics, exhibiting morphological properties of round-shape or spindle-shape; (d) has the ability to differentiate into mesoderm, endoderm and ectoderm derived cells; (e) providing human umbilical cord blood-derived adult stem cells having the property of differentiating into insulin-secreting pancreatic beta-like cells.

4. Differentiation of Cord Blood-derived Stem Cells into Pancreatic Beta-cells and Their Use

In the present invention, reference is made to known methods of differentiating embryonic stem cells from insulin-producing cells, but the cord blood-derived adult stem cells are differentiated into beta-like cells without selection of nestin positive cells. After 5-7 days, certain cells gather to form a pancreatic cell like structure (FIG. 2).

Nestin was originally known as a specific expression protein of neural stem cells, and has been known to express specifically in the brain and nervous system during development. Recently, however, they have been expressed in pancreas and ocular stem cells, and are currently used as markers of specific stem cells as well as neural stem cells.

Immunostaining results in detection of C-peptide (FIGS. 2A-a; red) and insulin (FIGS. 2A-b; green). Western blot analysis can be performed to determine the expression of C-peptide and Oct4. Pancreatic cell-like structures of the present invention show positive response in insulin and C-peptide staining.

Umbilical cord blood-derived adult stem cells (control) without treatment of differentiation medium did not show C-peptide expression but Oct4 expression. Compared with these controls, Western blot analysis showed that the insulin-producing cells of cord blood-derived adult stem cells. Shows successful differentiation. In addition, C-peptide expression excludes the possibility that insulin is taken up from the medium in these cells.

C-peptide is a useful indicator to know the insulin secretion ability of pancreatic β cells, has no hormonal action, and is measured by radioimmunoassay and expressed as C-peptide immunoreactivity (CRP). C-peptides also cross-react with proinsulins, but do not interfere with insulin antibodies, which is one of the useful tests in patients receiving insulin. The normal value of C-peptide is 1 to 2 ng / dL on an empty stomach and 4 to 6 ng / dL on sugar

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.

Umbilical cord blood-derived stem cells of the present invention can be used in a treatment protocol for diabetes that is enhanced, treated or replaced by engraftment, transplantation or infusion of stem cells or derived cell populations. The umbilical cord blood-derived stem cells of the present invention can replace or enhance insulin-producing cell tissue, resulting in new or changed tissue, or combined with biological tissue or structure. In addition, embryonic stem cells may be replaced with umbilical cord blood derived stem cells of the invention in a diabetes treatment protocol where typically embryonic stem cells are used.

In the present invention, only umbilical cord blood-derived stem cells are described in detail by way of example. Instead of umbilical cord blood-derived stem cells, adult stem cells derived from various human tissues such as bone marrow, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, and uterus may be used. It will be apparent to one of ordinary skill in the art.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

In particular, the following description and examples use the cord blood-derived stem cells, but can also be used in various human tissue-derived adult stem cells, such as bone marrow, cord blood, blood, liver, skin, gastrointestinal tract, placenta, and uterus.

All statistical analyzes were performed using SPSS version 13 software (SPSS Inc .; Chicago, IL, USA).

Example 1 Isolation and Culture of Adult Stem Cells from Umbilical Cord Blood

Human umbilical cord blood was obtained from umbilical vein immediately after normal delivery with maternal consent approved by Boramae Hospital Institutional Review Board (IRB), which was approved by the Seoul National University Institutional Review Board. Umbilical cord blood-monocytes (MNCs) were prepared from heparinized umbilical cord blood from single delivery by ficoll-hypaque density gradient centrifugation according to standard assays.

The cells obtained were supplemented with 10ng / ml of basic fibroblast growth factor (bFGF) [R & D Sytem Minneapolis, MN], 100U penicillin, 1000U streptomycin and 2mM / L glutamine (Gibco, grand island, NY), 20% FBS (Hyclone, Logan, UT) and DMEM complete medium (Dulbecco's Modified Eagle Medium) were added and about 3-5 × 10 ⁸ / ml of the cells were plated in a flask 75 cm ² and incubated for 3 days. After 3 days the suspension cells were transferred to a new 75 flask. After 7-10 days, the cells attached to the bottom of the flask merged to reveal several colonies. Three washes were followed by trypsinization to the attached cells and passaged into new vessels at a concentration of 10 ⁴ / mL. Every 7 days after incubation, appropriate amounts of cells were used for analysis.

After two weeks, once confluence and colony had formed, cells were passaged into the desired vessel. Once colonies are formed, the culture is ready for immuno-staining. When isolated insulin producing cells, colonies were removed by needle or trypsin treatment in a time-controlled fashion (3 or 5 minutes) to ensure that tightly attached spindle cells were not detached.

Example 2: Immunological Characteristics of Umbilical Cord Blood-derived Multipotent Adult Stem Cells

(1) Flow cytometry analysis of surface antigen expression

After separation, surface markers of UCB-derived colonies were analyzed by FACS antibodies. Cells were stained with fluorophores or phycoerythrin bound with CD4, CD20, CD29, CD34, CD44, CD45, CD90, CD105 and CD133 antibodies (all of Becton-Dickinson, San Jose, Calif., USA). Stained samples and unstained control cells were analyzed on a FACS Calibur analyzer, respectively.

As a result, as shown in Table 1 below, the cord blood-derived stem cells of the present invention were positive for CD29 (85%), CD45 (78.44%), CD44 (92.09%), and CD31 (90.53%). It was shown that the immunological properties of the negative, for all CD4, CD20, CD34, and CD51.

(2) immunohistochemistry

Umbilical cord blood-derived stem cells obtained in Example 1 were washed three times with PBS and fixed for 30 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, it is permeabilization for 10 minutes with PBS containing 0.1% Triton-X100 (Gibco). Rabbit anti-human Oct4 (Santa Cruz Co, USA) and mouse anti-human SSEA-4 (Santa Cruz Co, USA) as primary antibodies for 30 minutes at room temperature Used (1: 200). Red-Phycoerythrin bound to goat anti-rabbit IgG (Santa Cruz Co, USA) and fluorescent material bound to goth anti-mouse IgG were used as secondary antibodies.

As a result of Oct4 and SSEA4 double immunostaining, it was confirmed that adult umbilical cord blood-derived adult stem cells of the present invention express Oct4 and SSEA4 (FIG. 1).

Example 3: Differentiation of Umbilical Cord Blood-derived Stem Cells into Insulin-secreting Pancreatic Beta-like Cells

Colonies obtained by needle or trypsin treatment were obtained by centrifugation separating 0.1% trypsin (Gibco): 0.08% EDTA (sigma, Germany) (1: 1) in phosphate buffer (PBS, Gibco) for 1 minute. . The cells obtained for beta cell differentiation or FACS analysis were used. For differentiation into pancreatic beta cells (insulin producing cells), 20nM progesterone; 100 M putrescine; 1 g / mL laminin; 25 g / mL insulin; 30 nM sodium serenite; 10 mM nicotinamide (NA) (all Sigma); 50 g / mL transferrin; 5 g / mL fibronectin; B27 medium supplement (diluent 1:50) (both Gibco); And plated again in a poly-L-ornithine / laminin-coated tissue culture dish in "N2 medium + NA" containing DMEM / F12 (Gibco) supplemented with 15% FCS. After 24 hours the medium was exchanged and the cells were cultured in differentiation medium, with the difference that the cells were almost identical to "N2 medium + NA" and that DMEM / F12 (Gibco) was replaced with high concentration of DMEM (Gibco).

After 5 to 7 days of culture, as a result of immunostaining the cultured cells, it was confirmed that certain cells gather to form a pancreatic cell structure (FIG. 2C).

Example 4: Characterization of Insulin Secreting Pancreatic Beta-like Cells

For double immunostaining of insulin and C-peptide after fixation and permeabilization, as in the case of Oct4 and SSEA double immunostaining described above (Example 2), -Mouse anti-human insulin (abcam, MA, USA; 1: 100) was added to the culture as primary antibody. After overnight incubation at 4 ° C., goth anti-rabbit IgG bound phycoerythrin (PE) (Santa Cruz Co, USA) and goth anti-mouse IgG bound fluorescents were added as secondary antibodies. Immunostaining resulted in C-peptide (a; red in FIG. 2) and insulin (b in FIG. 2; green).

In addition, Western blot analysis was performed to determine the expression of C-peptide and Oct4. Pancreatic beta-cell-like structures were lysed and high salt buffer, 20 mM HEPES, pH 7.5; 25% glycerol; 0.42 M NaCl; 3 mM MgCl 2; 0.2 mM EDTA; 1 mM dithiothreitol; protein was extracted from protease inhibitor cocktail (Sigma; St. Louis, MO, USA). Proteins were separated on 15% SDS PAGE gels and transferred to nitrocellulose. C-peptide protein and Oct4 protein were detected with mouse monoclonal antibodies and chemiluminescent reagents (ECL; Amersham; Piscataway, NJ, USA).

The pancreatic cell-like structure of the present invention showed positive response in insulin and C-peptide staining, but in the control group not treated with C-peptide, no expression of Oct4 was observed (FIG. 3).

The present invention isolates adult stem cells from human umbilical cord blood and co-expresses insulin and C-peptide based on the fact that the stem cells express the embryonic stage specific marker SSEA-4 and the pluripotent stem cell marker Oct4. By providing a method for inducing an insulin-producing pancreatic islet-like structure, it has a very useful advantage in the treatment based on human umbilical cord blood derived stem cells for diabetes.

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 (10)

A method for differentiating adult stem cells into insulin-secreting pancreatic beta-like cells, wherein the adult stem cells derived from human tissue are cultured in a medium without nestin positive cells. The method according to claim 1, wherein the adult stem cells derived from human tissue, the mononuclear cells isolated from human tissue are cultured and recovered in a medium containing bFGF, penicillin, streptomycin, glutamine and FBS, and exhibits the following characteristics: How to feature: (a) has Oct4 and SSEA4 expression ability; (b) all show positive immunological properties for CD29, CD31, CD44, and CD45, and all show negative immunological properties for CD4, CD20, CD34, and CD51; (c) attached to and grown on plastics, exhibiting morphological properties of round-shape or spindle-shape; (d) has the ability to differentiate into mesoderm, endoderm and ectoderm derived cells; And (e) has the property of differentiating into insulin-secreting pancreatic beta-like cells. The method of claim 1, wherein the adult stem cells derived from human tissues are human tissue derived adult stem cells selected from the group consisting of breast, bone marrow, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, and uterus. The method of claim 3, wherein the adult stem cells derived from human tissues are adult stem cells derived from umbilical cord blood. The method for producing adult stem cells derived from human tissues having the following characteristics, characterized in that the mononuclear cells isolated from human tissues are cultured in a medium containing bFGF, penicillin, streptomycin, glutamine and FBS, and then recovered. (a) has Oct4 and SSEA4 expression ability; (b) all show positive immunological properties for CD29, CD31, CD44, and CD45, and all show negative immunological properties for CD4, CD20, CD34, and CD51; (c) attached to and grown on plastics, exhibiting morphological properties of round-shape or spindle-shape; (d) has the ability to differentiate into mesoderm, endoderm and ectoderm derived cells; And (e) has the property of differentiating into insulin-secreting pancreatic beta-like cells. 6. The adult stem cell of claim 5, wherein the adult stem cell derived from human tissue is derived from umbilical cord blood. An adult stem cell derived from a human tissue obtained by the method of claim 5 or 6 and having the following characteristics: (a) has Oct4 and SSEA4 expression ability; (b) all show positive immunological properties for CD29, CD31, CD44, and CD45, and all show negative immunological properties for CD4, CD20, CD34, and CD51; (c) attached to and grown on plastics, exhibiting morphological properties of round-shape or spindle-shape; (d) has the ability to differentiate into mesoderm, endoderm and ectoderm derived cells; And (e) has the property of differentiating into insulin-secreting pancreatic beta-like cells. An insulin-secreting pancreatic beta-like cell which is differentiated by the method of any one of claims 1 to 4 and exhibits the property of expressing insulin and C-peptide but not Oct4. A cell therapeutic agent for the treatment of diabetes, comprising as an active ingredient human stem cells derived from claim 7 having the characteristics of differentiating into insulin-secreting pancreatic beta-like cells. Cell therapy for the treatment of diabetes comprising the insulin-secreting pancreatic beta-like cells of claim 8 as an active ingredient.

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