JP2011139691A - Method for producing multipotent stem cell originated from amnion - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a cell population in which the proportion of multipotent stem cells free from tumorigenicity and differentiated to all cell lines is increased. <P>SOLUTION: The method for producing the multipotent stem cell originated from amnion includes: a step of separating an amnion tissue from a placenta tissue after parturition of a mammal; a step of culturing the amnion tissue, and preparing cells originated from the amnion, and comprising single cell suspended matter; a step of bringing the cells originated from the amnion into contact with one or more kinds of antibodies selected from the group consisting of anti-ABCG1 antibody, anti-SLC21A12 antibody, anti-CLDN3 antibody, anti-SCNN1A antibody, anti-TMPRSS4 antibody, anti-CLDN4 antibody, anti-CLDN7 antibody, anti-EZR antibody, anti-ST14 antibody and anti-MUC18 antibody; and a step of selecting cells binding with the antibodies. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for preparing amniotic membrane-derived cells from an amnion derived from a mammalian fetus, isolating, culturing and storing pluripotent stem cells from the cell population, and therapeutic use thereof.

  Embryonic stem (ES) cells established from the inner cell mass (ICM) of mammalian blastocyst stage fertilized eggs have pluripotency and can differentiate into all cell lineages. A technique for inducing differentiation into a specific cell line in vitro using ES cells has been developed, and application to regenerative medicine in the form of cell transplantation or organ transplantation is expected. In addition, if ES cells having pluripotency are injected into the lumen of a blastocyst stage fertilized egg, the producing individual forms a chimeric mouse, and a knockout mouse lacking a specific gene is produced using this technique. be able to.

  Yamanaka et al. Introduced 4 genes highly expressed in ES cells, Oct4, Sox2, Klf4 and c-Myc into somatic cells to overexpress them, so that somatic cells can be initialized to an ES-like state. (For example, refer patent document 1). Induced pluripotent stem cells (iPS cells) produced by this method can be induced to differentiate into all cells in the same manner as ES cells. Genes introduced using retroviral vectors to generate iPS cells are incorporated into chromosomal DNA and cause ectopic expression. Therefore, tumor formation occurs in about 20% of chimeric animals generated using iPS cells. Was recognized. Since then, improved methods have been developed that do not cause recombination into chromosomal DNA using adenoviral vectors or plasmid vectors instead of retroviral vectors, but all have the problem of low somatic cell initialization efficiency. . However, since iPS cells can be prepared from the patient's own cells, they are advantageous over ES cells in terms of immunocompatibility during transplantation.

  On the other hand, adult stem cells do not spontaneously differentiate but can be induced to differentiate by applying appropriate growth conditions. However, adult stem cells are not immortal and most of them have the disadvantage that they lose their differentiation ability after a certain number of passages in culture. It has been reported that certain pluripotent stem cells can be isolated from fetal-derived cells present in amnion tissue. For example, in Patent Document 2, the expression of Oct4 gene, Sox2 gene, FGF4 gene and Rex1 gene is observed in RT-PCR, isolated from human amnion mesenchymal cell layer, and vimentin positive and CK19 positive in immune cell staining It is disclosed that the side population cells can differentiate into at least neuronal cells. In Patent Document 3, cells isolated from amniotic tissue are cultured in Dulbecco's modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS) to prepare amnion-derived stem cells consisting of a single cell culture. ing. The amnion-derived stem cells isolated in this way are CD29 and CD90 positive as cell surface markers and CD45 and CD11b negative. In addition, it is described that such amnion-derived stem cells can be induced to differentiate into nerve, bone, fat, liver and heart cells by culturing under appropriate conditions.

International Publication WO2007 / 069666 Pamphlet JP2004-254682 International Publication WO2008 / 003042 Pamphlet

  However, ES cells and iPS cells tend to spontaneously differentiate into various types of tissues, and when injected into immunodeficient mice, teratomas are known to differentiate into multiple tissue types. In addition, when cells produced by inducing differentiation of ES cells or iPS cells are transplanted into a patient, there is a risk that a tumor will be generated at a certain rate due to the presence of undifferentiated cells to be mixed.

  Amnion-derived stem cells maintain a higher proliferative capacity than adult stem cells and can retain their pluripotency for longer in culture, but it is not always clear whether they can induce differentiation into all cells. For example, there is no report that it can differentiate into hematopoietic cells. The object of the present invention is to solve these problems of the prior art.

  The present invention has been made in view of the above problems, and a cell population in which the ratio of pluripotent stem cells is increased using a specific cell surface marker for amnion-derived cells prepared from a postpartum placental tissue of a mammal It was found that can be acquired selectively. Cells contained in the obtained cell population were confirmed by immunostaining to express the nuclear reprogramming factors Oct3 / 4, Sox2 and Klf4, and in the same manner as ES cells and iPS cells. It was found to be a pluripotent stem cell that can differentiate into all somatic cells.

  That is, in the first aspect of the present invention, a step of separating the amniotic membrane from the postpartum placental tissue of a mammal, a step of culturing the amniotic membrane and preparing an amniotic membrane-derived cell comprising a single cell suspension, and the amniotic membrane Any one selected from the group consisting of anti-ABCG1 antibody, anti-SLC21A12 antibody, anti-CLDN3 antibody, anti-SCNN1A antibody, anti-TMPRSS4 antibody, anti-CLDN4 antibody, anti-CLDN7 antibody, anti-EZR antibody, anti-ST14 antibody and anti-MUC18 antibody There is provided a method for producing a cell population in which the ratio of pluripotent stem cells is increased, comprising the step of contacting with one or more antibodies and the step of selecting cells that bind to the antibodies.

  The amnion-derived cells are obtained by preparing maternal serum from a clot attached to the postpartum placental tissue and culturing the amnion and cells that migrate from the medium in a medium containing the maternal serum. Is preferred.

  In another aspect of the present invention, there is provided a cell population produced by the above method, which expresses genes for Oct3 / 4, Sox2 and Klf4, and has no tumorigenicity when transplanted into an immunodeficient animal. A cell population comprising potent stem cells is provided. This cell population can be used as a pluripotent stem cell composition for transplantation therapy or a pharmaceutical composition for use in cell therapy.

  In still another aspect of the present invention, a step of isolating a clot and an amniotic membrane from human postpartum placental tissue and its attachment, a step of preparing maternal serum from the clot, and the amniotic membrane of the maternal body A method for culturing amnion-derived pluripotent stem cells, comprising the steps of: culturing in a medium containing serum derived but not serum derived from a heterologous animal to prepare amnion-derived cells comprising a single cell suspension.

  Furthermore, in a different viewpoint, a protective agent is added to the step of separating the amniotic membrane from human postpartum placental tissue, the step of chopping the amniotic membrane and treating with collagenase, the amniotic strip and cells migrating therefrom. A method of preserving amnion-derived pluripotent stem cells, comprising the step of freezing. The outline of these inventions does not enumerate all the necessary features, and therefore a sub-concept of these feature groups can also be an invention.

  According to the production method of the present invention, pluripotent stem cells can be isolated easily and efficiently from amnion-derived cells prepared from a postpartum placenta. Since the stem cells produced by such a method do not have tumorigenicity, they can be transplanted to a living body as they are or after being induced to differentiate into desired target cells.

In an Example, it is a figure which shows the result of FACS about the cell isolate | separated and cultured from the human amniotic membrane. It is a figure which shows the result of having carried out the immuno-staining of the human amnion primary culture cell with the anti-ABCG1 antibody. It is a figure which shows the result of having immunostained the human amnion primary culture cell with three types of antibodies, anti-human Oct4 antibody, anti-human Sox2 antibody, and anti-human Klf4 antibody.

(1) Acquisition of amnion-derived cells from mammals In this specification, “mammal” includes cattle, horses, dogs, guinea pigs, mice, rats and the like in addition to primates including humans.
The amniotic membrane-derived cells according to the present invention can be easily isolated from the postpartum placental tissue of the mammal by using techniques known to those skilled in the art. In the present specification, “postpartum placental tissue” refers to a tissue delivered with a fetus at the time of delivery of a mammal, and includes amniotic membrane, chorion, placental lobe and the like. The term “amniotic membrane” is the membrane closest to the fetus among the two membranes covering the fetus together with the chorion, in which it fills the amniotic fluid and the fetus floats in the amniotic fluid. In the present invention, “amniotic membrane” and “amniotic tissue” are used interchangeably.

  In one embodiment, mammalian postpartum placental tissue is isolated from cells migrating from the amniotic membrane by mechanical treatment, treatment with digestive enzymes, treatment with chelating agents, or combinations thereof, It can be obtained as a cell suspension. The chelating agent weakens the binding between adjacent cells, disperses the tissue, and separates individual cells without appreciable cell damage. Further, enzymatic separation between cells can be achieved by finely chopping tissue and treating the minced tissue with one digestive enzyme alone or in combination with several digestive enzymes. Examples of the digestive enzyme include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase and / or hyaluronidase, DNase, and pronase. In addition, mechanical destruction of the placental tissue of mammals can be performed using, for example, a pulverizer, a blender, a sieve, a homogenizer, a cell compressor, or an ultrasonic processor.

  In the present invention, as a method for isolating amnion-derived cells from mammalian placenta tissue, a method using collagenase among the digestive enzyme treatments is more preferable. For example, incubation is performed at 37 ° C. for 30 minutes to several hours, preferably about 1 hour, using collagenase at a concentration of 0.5 to 5 mg / ml.

  The amnion tissue thus fragmented and the amnion-derived cells obtained by enzymatic treatment thereof can be stably preserved for a long period of time by adding a preservative, particularly a cryoprotectant, and cryopreserving them. Cell cryoprotectants include water-soluble polymers such as dextran, hydroxyethyl starch, and polyvinylpyrrolidone known as extracellular cryoprotectants, and low molecular weight organic compounds such as dimethyl sulfoxide and glycerol known as intracellular cryoprotectants. However, the present invention is not limited to these and may be selected as appropriate. In addition, in the production of the frozen product according to the present invention, a cell membrane protective agent, a nutrient component, and the like can be added as necessary in addition to the cryoprotective agent.

  In freezing and storing such a composition, a simple method using a deep freezer at minus 80 ° C. or a method of slowly cooling it with a program freezer and storing it in liquid nitrogen can be used. The cell frozen material thus obtained has extremely high cell viability when culturing amniotic cells after thawing after storage for a long period of time, compared with the case where the amniotic tissue is frozen as it is. Suitable for storage as an amniotic cell bank. Since the cell frozen material stored in this manner contains fetal pluripotent stem cells, this allows not only the public cell bank for others but also the private cell bank for the fetus itself and its mother, Or it is useful as a private cell bank for families.

(2) Culture of amnion-derived cells The amnion-derived cells isolated as described above can be cultured by a known method known in the art. Specifically, it is preferable to perform culture by suspending in Dulbecco's modified Eagle medium (DMEM, low glucose, Sigma) + 10% FBS. In addition, when culturing human amnion-derived cells, maternal serum is prepared from a clot attached to human postpartum placental tissue, and contains the maternal serum but substantially does not contain serum from a different animal. It is preferable to culture in a medium. By not containing sera derived from different animals, contamination of infectious substances such as viruses derived from these is prevented, and the stem cell composition can be transferred to individuals by using sera from the genetically closest individuals. This is because the contamination with immunogenic substances during transplantation is also reduced.

(3) Isolation and enrichment of pluripotent stem cells using antibodies against specific cell surface markers Several methods have already been disclosed as methods for isolating stem cells from amnion-derived cells. For example, Hoechst 33342 (Hoechst33342) is a fluorescent dye incorporated into the AT sequence of DNA, and has high membrane permeability and stains cells alive. Among the cell populations stained with this dye, those with a relatively low degree of staining are called SP (side population) cells, which initially contain hematopoietic stem cells in high frequency. It was revealed. Thereafter, it has been reported that stem cells can be similarly obtained from amnion-derived cells (Patent Document 2). The mechanism by which SP cells appear is that pigments are excreted by certain pumps such as MDR (multi drug resistance gene), which is a multidrug resistance gene that they express. Interestingly, such properties are common to organ stem cells such as liver, kidney, and muscle, and can be applied to a method for identifying organ stem cells without using antibodies. Amniotic membrane-derived cells cultured by the above method contained only 1% or less low frequency SP cells.

  When analyzing the surface markers of these amnion-derived SP cells, as shown in the examples described later, the expression of ABCG1, SLC21A12, CLDN3, SCNN1A, TMPRSS4, CLDN4, CLDN7, EZR, ST14, and MUC18 gene is enhanced. Cells expressing these genes on the cell surface and in the cells were detected. Among these gene products, ABCG1 is the mammalian homologue of the Drosophila White Gene. ABCG1 cDNA is cloned from mouse and human macrophage cell lines and encodes a putative protein of approximately 70 kDa. In human macrophages, two types of apparent molecular weights of about 60 kDa and about 110 kDa have been reported. ABCG1 plays an important role in transporting excess cholesterol to HDL in macrophages. It is also involved in the transport of choline-containing phospholipids (phosphatidylcholine, sphingomyelin). ABCG1 in the cell membrane has an N-terminal cytoplasmic ATP-binding region and a six-transmembrane helix structure on the C-terminal side. The ABCG1 protein differs from the MDR (ABCB1) transporter, which has a 12-transmembrane structure and exhibits a wide range of substrate specificity, in that it forms a homodimer and is expressed and functions on the cell membrane.

SLC21A12 is a member of the transporter (solute transporter) family that transports substrates using an ion concentration gradient, and includes SLCO4A1 (Solute carrier organic anion transporter).
Also called family member 4A1). SLC21A12 protein expressed in human placenta has been reported to be involved in membrane transport of sulfated steroids. The claudin family (CLD3, 4, 7) proteins are transmembrane proteins that are directly involved in cell adhesion and tight junction formation. SCNN1A is a subunit of a channel-forming protein called sodium channel / non-voltage-dependent 1α. It has been reported that mutations in this gene are associated with type I pseudohypoaldosterosis. TMPRSS4 is a membrane-bound serine protease. EZR (Ezrin) is a protein that binds to the membrane surface on the cytoplasm side and functions as a substrate for tyrosine kinase in microvilli. It is located between the cell membrane and the actin cytoskeleton and plays a role in cell surface structure adhesion, migration, and organization. ST14 is an epithelial and integral membrane protein serine protease that activates other protease precursors and latent growth factors. MUC18 is a cell surface protein also called MCAM (melanoma cell adhesion molecule) or CD146.

  Therefore, the present invention uses an antibody having a specific affinity for the cell surface marker to separate / recover cells in which the marker protein is expressed on the cell membrane surface. Any one selected from the group consisting of anti-ABCG1 antibody, anti-SLC21A12 antibody, anti-CLDN3 antibody, anti-SCNN1A antibody, anti-TMPRSS4 antibody, anti-CLDN4 antibody, anti-CLDN7 antibody, anti-EZR antibody, anti-ST14 antibody, and anti-MUC18 antibody Contacting with one or more antibodies, and selecting cells that bind to the antibodies. For example, a method using antibody-bound beads, affinity chromatography, a panning method using an antibody attached to a solid matrix such as a plate, or a cell sorter can be used. For example, the target cells are automatically separated and collected by labeling the antibody with an appropriate dye or fluorescent substance and using a fluorescence activated cell sorter (FACS). A fluorescently labeled secondary antibody may be used for labeling the antibody. Dead cells can be removed by selection with a dye associated with dead cells (propidium iodide: PI).

  In a preferred embodiment, the antibody is any antibody specific for ABCG1 protein or a fragment thereof. The antibodies used in the present invention retain sufficient specificity to specifically bind to the ABCG1 protein or fragment thereof and are either natural or recombinant, either synthetic or naturally derived, monoclonal or polyclonal. Any antibody or fragment thereof is included.

Conjugate the antibody with other suitable molecules and compounds including enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, chromatographic resins, solid supports or drugs. )can do. Enzymes that can be conjugated to antibodies include alkaline phosphatase, peroxidase, urease and β-galactosidase. Fluorescent dyes that can be conjugated to the antibody include fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanin and Texas Red. Metal compounds that can be conjugated to the antibody include ferritin, colloidal gold, and particularly colloidal superparamagnetic beads. Haptens that can be conjugated to antibodies include biotin, digoxigenin, oxazalone and nitrophenol. Radioactive compounds that can be conjugated or incorporated into antibodies are known in the art and include any of technetium 99m, ¹²⁵ I, and including but not limited to ¹⁴ C, ³ H and ³⁵ S. An amino acid containing such a radionuclide can be mentioned.

  The cell surface marker including ABCG1 protein or a fragment thereof is not particularly limited as long as it is derived from mammals, but in addition to primates including humans, proteins derived from cattle, horses, dogs, guinea pigs, mice and rats Is preferred. With the appropriate antigen protein, the antibody or functional part thereof can be obtained by methods known in the art. Such methods include, but are not limited to, isolating B cells with cell surface antibodies of the desired specificity, cloning DNA expressing light and heavy chain variable regions, and appropriate Expression of the recombinant gene in a suitable host cell. Standard monoclonal antibody production techniques can be used when the antibodies are obtained from immortalized antibody-producing hybridoma cells. These hybridomas immunize animals with cell surface markers including the above-mentioned ABCG1 protein or fragments thereof, and preferably convert B lymphocytes isolated from the immunized host spleen into compatible immortalized cells, preferably It can be produced by fusing with B cell myeloma.

(4) Properties of pluripotent stem cells “Detection” or “quantification” of gene expression (eg, mRNA expression, polypeptide expression) in cells obtained by the method of the present invention includes, for example, It can be performed by an immunological measurement method or the like. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of the immunological measurement method include an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method. Examples of the quantitative method include an ELISA method and an RIA method. It can also be performed by a gene analysis method using an array (for example, a DNA array, a protein array, etc.). DNA array analysis is extensively outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and latest PCR method"). Examples of gene expression analysis methods include, but are not limited to, RT-PCR method, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation method, and CAGE method.

  As a result of gene expression analysis by the DNA microarray and immunostaining method, at least three genes, Oct3 / 4, Sox2 and Klf4, which are nuclear reprogramming factors, are expressed in the amnion-derived stem cells produced by the method of the present invention Became clear. These genes encode transcription factors that can initialize the state of differentiation of cells and are characteristically expressed in pluripotent stem cells that can differentiate into all cell lines. Therefore, it is suggested that the amniotic membrane-derived stem cells produced by the method of the present invention are natural pluripotent stem cells and iPS-like cells.

(5) Pluripotent stem cell composition for transplantation treatment The amnion-derived pluripotent stem cell population produced by the method of the present invention can be used as a composition for transplantation treatment. The transplantation treatment is prevention or treatment of a disease accompanied by tissue destruction, for example, neurological disease, respiratory disease, circulatory disease, liver disease, pancreatic disease, digestive system disease, kidney disease, skin disease. And applicable to lung diseases and the like. That is, the amniotic membrane-derived pluripotent stem cell population of the present invention is washed with physiological saline using an apparatus capable of concentrating, washing, and recovering cells, and removes antibodies, cytokines, etc. used for culture and separation as much as possible. It is preferable to do. The stem cell population washed and concentrated in this way can be used for the prevention or treatment of a disease accompanied by tissue destruction by injecting it into a vein by a normal infusion method or directly injecting it into a diseased site. The dose of the stem cell population varies depending on the disease or tissue destruction, but it is preferable to administer 10 to 10 ⁶ cells per time.

  The present invention also provides cells induced to differentiate from the above-mentioned amnion-derived pluripotent stem cell population. Although it does not specifically limit as a differentiated cell, For example, an osteoblast, a nervous system cell, a chondroblast, a hepatocyte, a pancreatic cell, a blood cell, or a brain cell etc. are mentioned. Neural cells include neural progenitor cells, neural stem cells that differentiate from the neural progenitor cells, neurons (for example, motor neurons or dopaminergic neurons), astrocytes, oligodendrocytes, or Schwann cells. Etc. Further, osteoprogenitor cells that differentiate into osteoblasts and the like can also be mentioned.

  The method for inducing differentiation from the above-mentioned amnion-derived pluripotent stem cell population to the desired cells as described above is not particularly limited, and a known method may be used. For example, the method of processing using a well-known cell differentiation inducer is mentioned. Examples of the cell differentiation inducing factor include bone morphogenetic factor; neurotrophic factor; factor belonging to TGF-β superfamily such as tumor growth factor (TGF) -β or activin; basic fibroblast growth factor (bFGF) or acidic fiber Factors belonging to the FGF superfamily, such as blast growth factor (aFGF); factors such as leukemia inhibitory factor (LIF), preferably bone morphogenetic factor or neurotrophic factor. BMPs such as BMP-2, -4, -5, -6, -7, -8, -9, -10, -11, and -12 are proteins that promote bone formation and cartilage formation. Family, especially BMP-2, -4, -6, -7. Examples of the neurotrophic factor include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), glial-derived neurotrophic factor (GDNF), NT-4 / 5 and the like. The NGF family is preferable.

  Furthermore, the human amnion-derived pluripotent stem cell population according to the present invention can be applied as a medicine. Specifically, the amnion-derived pluripotent stem cell population is preferably used as a cell or organ regeneration agent. The “cell” in this case is not particularly limited, and examples thereof include osteoblasts, various physiologically active substance-producing cells, and nervous system cells. Examples of the physiologically active substance-producing cells include dopamine-producing cells, and examples of the nervous system cells include, but are not limited to, motor neurons. Neural cells also include neural progenitor cells; neural stem cells; neurons, particularly dopaminergic neurons; astrocytes; oligodendrocytes; or Schwann cells.

  In addition, cells induced to differentiate from the amnion-derived pluripotent stem cell population according to the present invention can also be applied as pharmaceuticals. For example, when the amnion-derived pluripotent stem cell population according to the present invention is induced to differentiate into cells suitable for a desired tissue or organ, the differentiation-induced cell cures or alleviates or damages the tissue or organ. It can be used as a medicament for prevention of deterioration. More specifically, an amnion-derived pluripotent stem cell population according to the present invention or a cell that is differentiated from the amnion-derived pluripotent stem cell population and that is compatible with a desired tissue or organ is transplanted into a damaged tissue or organ. By doing so, the tissue or organ can be regenerated. Tissues or organs that can be regenerated in this way are not particularly limited, for example, tissues such as blood vessels, cornea, meniscus, brain tissue, skin, subcutaneous tissue, epithelial tissue, bone tissue or muscle tissue; or eye, Examples include organs such as lung, kidney, heart, liver, pancreas, spleen, digestive tract including small intestine, bladder, ovary or testis. Examples of the tissue also include neural progenitor cells; neural stem cells; neurons, particularly motor or dopaminergic neurons; astrocytes; oligodendrocytes or Schwann cells. In particular, the amniotic membrane-derived pluripotent stem cell population according to the present invention or osteoblasts induced to differentiate from the amnion-derived pluripotent stem cell population is preferably used as a prophylactic or therapeutic agent for osteoporosis or an osteogenic agent. An amnion-derived pluripotent stem cell population according to the present invention, a neural progenitor cell differentiated from the amnion-derived pluripotent stem cell population, or a dopaminergic neuron further induced to differentiate from the neural progenitor cell is Parkinson It is preferably used as a prophylactic / therapeutic agent for diseases.

  In the medicament according to the present invention as described above, the major histocompatibility antigen (MHC) of the amnion-derived pluripotent stem cell population according to the present invention is preferably specified. If MHC is clearly specified in this way, rejection at the time of cell transplantation into a recipient having the same type of MHC as the amnion donor can be reduced. That is, the present invention relates to rejection at the time of cell transplantation characterized by transplanting a mammalian amnion-derived pluripotent stem cell population having the same type of MHC as the recipient's MHC or cells induced to differentiate from the stem cells. Methods of treatment with reduced response are provided.

  The medicament according to the present invention as described above may be an amnion-derived pluripotent stem cell population according to the present invention which is an active ingredient or a cell itself induced to differentiate from the amnion-derived pluripotent stem cell population, Manufactured by mixing the active ingredient and a pharmacologically acceptable carrier according to a method known per se [a method commonly used in the field of pharmaceutical technology, such as the method described in Japanese Pharmacopoeia (for example, the 13th revision)]. Is done. As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. For example, solvents, solubilizers, suspending agents, isotonic agents, buffering agents, painless agents in liquid preparations. And the like. If necessary, formulation additives such as surfactants, foaming agents, dyes, sour agents, preservatives, antioxidants, colorants, sweeteners, and corrigents can also be used.

  Examples of pharmaceutical dosage forms according to the present invention include injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), external preparations (eg, nasal preparations, transdermal) Preparations, ointments, etc.), suppositories (eg, rectal suppositories, vaginal suppositories, etc.), pellets, drops, sustained release preparations (eg, sustained release microcapsules, etc.) and the like. The dosage of the pharmaceutical agent according to the present invention varies depending on the pharmaceutical dosage form, the type of pathological condition to be treated, the symptoms and the severity of the disease, the patient's age, sex or weight, the administration method, etc. However, the doctor can make a decision by comprehensively judging the above situation. The administration route of the medicament according to the present invention is not particularly limited. For example, when the medicament according to the present invention is an injection, for example, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection or intraperitoneal injection is used. Examples of such medically suitable dosage forms can be exemplified.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the following examples.

(1) Method for culturing amniotic cells Human amnion from natural labor was collected, and the amniotic epithelium and chorion were minced with scissors. The amnion strips were treated with 2 mg / ml collagenase at 37 ° C. for 1 hour. Collagenase-treated amnion strips and cells that migrated therefrom were collected, washed three times with phosphate buffered saline (PBS), seeded in a flask, and 10% fetal bovine serum (DMEM) in Dulbecco's modified Eagle medium (DMEM). Cultured and passaged for 1 month in medium supplemented with BSA).

(2) Method of SP fractionation and non-SP fractionation Cells that spread almost completely over the adhesion surface of the flask were treated with 0.25% trypsin-EDTA (Invitrogen) at 37 ° C. for 10 minutes to collect the cells. The obtained cells were suspended in PBS and mixed well, then Hoechst 33342 was added to a final concentration of 2.5 μg / ml, and staining was performed at 37 ° C. for 30 minutes. Excitation was performed with a 350 nm UV laser using a fluorescence activated cell sorter (Becton Dickinson Co., Ltd.) to detect Hoechst Blue (350 nm) and Hoechst Red (675 nm). The result is shown in FIG. SP cells present at a ratio of 0.42% in the lower left part of the graph, that is, in the region where the fluorescence intensities at both wavelengths were weak were collected. This SP cell fraction was not completely lost even when verapamil, an MDR molecule function inhibitor, was added. Since at least 2.0 × 10 ⁸ primary cultured cells were obtained from one amniotic membrane, it was considered possible to separate at least 0.8 × 10 ⁶ SP cells.

(3) DNA chip analysis:
Total RNA was extracted from each of the SP and non-SP fraction cells using Trizol (Invitrogen). For each cell, 20 ng of total RNA was used to synthesize c-RNA using a two cycle c-RNA synthesis kit (Affymetrix). Subsequently, gene expression analysis was performed with an HG-U133A chip (Affymetrix) using the c-RNA of each cell. The chip is read using GeneArray Scanner (Affymetrix), and the gene expression level is targeted using Microarray Suite 5.0, which is Affymetrix software.
global with intensity set to 1000
Analysis was performed by a scaling method (relative expression level when the average expression level of all genes was 1000). As a result, 10 genes including the following ATP transporter ABCG1 gene were found as genes of cell surface localized proteins whose expression was increased about 3 times or more in SP fraction cells compared to non-SP fraction cells. The expression levels of these genes by chip analysis are as follows.

(4) Cell staining analysis:
Primary cultured amniotic cells were seeded on a cover glass, cultured for 24 hours, and then fixed by incubating with PBS containing 2% paraformaldehyde for 30 minutes on ice. Subsequently, the plate was incubated with PBS containing 0.1% Triton X-100 (polyoxyethylene (10) octylphenyl ether) for 10 minutes at room temperature, followed by membrane permeabilization, followed by blocking Buffer (2% Normal Swine Serum / 0.05). %
The solution was changed to TritonX-100 in PBS, and incubated at room temperature for 30 minutes to perform a blocking reaction. Then, it was incubated overnight at 4 ° C. using an anti-human ABCG1 rabbit polyclonal antibody (Santa Cruz Biotechnology, # sc-11150). After removing the primary antibody, it was incubated with an anti-rabbit IgG (H + L) antibody (Invitrogen, # A-31572) conjugated with Alexa555 as a secondary antibody at room temperature for 1 hour. Subsequently, DAPI (2 μg / ml) was added and treated at room temperature for 15 minutes to label the nucleic acid. The immunostained cells were analyzed using a confocal laser microscope (ZEISS). The result is shown in FIG. Cells in which the ABCG1 protein shown in red was localized on the cell surface and in the cytoplasm were found. Considering the result of the above DNA chip analysis, it is considered that cells expressing ABCG1 on the cell surface more frequently exist in SP cells.

  Next, three types of antibodies are used as primary antibodies: anti-human Oct4 rabbit polyclonal antibody (abcam, # ab19857), anti-human Sox2 goat polyclonal antibody (R & D systems, # AF2018) and anti-human Klf4 mouse monoclonal antibody (abcam, # ab75486). Was used to stain primary cultured amniotic cells. As secondary antibodies, anti-goat IgG (H + L) antibody conjugated with Alexa555 (Invitrogen, # A21432), anti-rabbit IgG (H + L) antibody conjugated with Alexa647 (Invitrogen, # A-21245), and Alexa488 were conjugated. Anti-mouse IgG (H + L) antibody (Invitrogen, # A21202) was used. The result taken with a confocal laser microscope is shown in FIG. Among these cells, there are cells in which Oct4 shown in purple, Sox2 shown in red, and Klf4 shown in green are simultaneously expressed. Therefore, amnion-derived SP cells and / or ABCG1 are expressed as cells. The cell fraction separated as a surface marker expresses three transcription factors necessary for cell nuclear reprogramming, suggesting that iPS-like pluripotent stem cells are included.

  As described above, the present invention provides a method for producing a cell fraction in which the proportion of pluripotent stem cells is increased by using an antibody against a marker protein expressed on the surface of an amnion-derived stem cell, thereby realizing regenerative medicine. It is thought that this is a useful technology for

Claims (7)

Separating the amniotic membrane from the postpartum placental tissue of the mammal;
Culturing the amnion and preparing amnion-derived cells comprising a single cell suspension;
The amnion-derived cells are selected from the group consisting of anti-ABCG1 antibody, anti-SLC21A12 antibody, anti-CLDN3 antibody, anti-SCNN1A antibody, anti-TMPRSS4 antibody, anti-CLDN4 antibody, anti-CLDN7 antibody, anti-EZR antibody, anti-ST14 antibody and anti-MUC18 antibody. Contacting with one or more antibodies,
Selecting cells that bind to the antibody;
A method for producing a cell population in which the proportion of pluripotent stem cells is increased.   The amnion-derived cells are obtained by preparing maternal serum from a clot attached to the postpartum placental tissue and culturing the amnion and cells that migrate from the medium in a medium containing the maternal serum. The method of claim 1.   A pluripotent cell population produced by the method according to claim 1 or 2, which expresses genes of Oct3 / 4, Sox2 and Klf4, and has no tumorigenicity when transplanted to an immunodeficient animal. A cell population containing sex stem cells.   A pluripotent stem cell composition for transplantation treatment comprising the cell population according to claim 3.   A pharmaceutical composition for use in cell therapy comprising the cell population of claim 3 and a pharmaceutically acceptable carrier. Isolating clots and amniotic membrane from human postpartum placental tissue and its deposits, respectively;
Preparing maternal serum from the clot;
Cultivating the amniotic membrane in a medium containing the maternal serum but not from a heterologous animal, and preparing amnion-derived cells consisting of a single cell suspension. A method for culturing sex stem cells. Separating the amniotic membrane from human postpartum placental tissue;
Chopping the amniotic membrane and treating with collagenase;
Adding a protective agent to the amnion strips and cells migrating therefrom and freezing;
A method for preserving amnion-derived pluripotent stem cells, comprising:

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