WO2013162330A1 - Population de cellules souches mésenchymateuses chimériques et son procédé de préparation, et procédé de production de parathormones à l'aide de cellules souches dérivées d'amygdales - Google Patents

Population de cellules souches mésenchymateuses chimériques et son procédé de préparation, et procédé de production de parathormones à l'aide de cellules souches dérivées d'amygdales Download PDF

Info

Publication number
WO2013162330A1
WO2013162330A1 PCT/KR2013/003644 KR2013003644W WO2013162330A1 WO 2013162330 A1 WO2013162330 A1 WO 2013162330A1 KR 2013003644 W KR2013003644 W KR 2013003644W WO 2013162330 A1 WO2013162330 A1 WO 2013162330A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
mesenchymal stem
chimeric
stem cell
parathyroid
Prior art date
Application number
PCT/KR2013/003644
Other languages
English (en)
Korean (ko)
Inventor
김한수
우소연
유경하
조경아
조인호
박윤신
Original Assignee
이화여자대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR20120044970A external-priority patent/KR101508413B1/ko
Priority claimed from KR1020130045033A external-priority patent/KR101520531B1/ko
Application filed by 이화여자대학교 산학협력단 filed Critical 이화여자대학교 산학협력단
Publication of WO2013162330A1 publication Critical patent/WO2013162330A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution

Definitions

  • the present invention is a method for producing a chimeric mesenchymal stem cell group comprising the step of mixed culture of tonsil-derived mesenchymal stem cells obtained from a separate donor, chimeric mesenchymal stem cell group prepared by the method, parathyroid gland from tonsil stem cells A method for inducing differentiation of tissue cells and a method for producing parathyroid hormone from parathyroid tissue cells obtained by the method.
  • human tonsils are a type of lymphatic epithelial tissue that exists in the oropharyngeal nasopharynx of the human body, and performs its immune function until puberty and then gradually degenerates, causing the tonsils to become abnormally enlarged (adenotonsillar hyperplasia). Infections such as tonsillitis and tonsillitis can be removed by tonsillectomy.
  • Parathyroid hormone is a hormone composed of 84 amino acids secreted from the parathyroid gland, and is a major hormone that controls calcium concentration in vivo, and can be used as a therapeutic agent for parathyroid hypoplasia and osteoporosis. Increasingly, however, no method has yet been developed to effectively produce parathyroid hormone.
  • the present inventors have made diligent efforts to improve the utility of stem cells derived from tonsils, resulting in tonsil tissues obtained from a single donor even if mixed cultures of stem cells derived from different donors are cultured.
  • the stem cell group can be used for stem cell banks, and also that parathyroid tissue cells can be differentiated from tonsil stem cells, thereby confirming that parathyroid hormone is normally produced and secreted, thereby completing the present invention.
  • One object of the present invention is to provide a method for producing a chimeric mesenchymal stem cell population comprising a mixed culture of tonsil-derived mesenchymal stem cells obtained from separate donors.
  • Another object of the present invention is to provide a chimeric mesenchymal stem cell group produced by the above method.
  • Still another object of the present invention is to provide a method for producing a chimeric mesenchymal stem cell group having improved proliferative capacity, including freezing and thawing the chimeric mesenchymal stem cell group.
  • Still another object of the present invention is to provide a chimeric mesenchymal stem cell group having improved proliferative capacity prepared by the above method.
  • Still another object of the present invention is to provide a method for differentiating the chimeric mesenchymal stem cell population.
  • Still another object of the present invention is to provide a cell therapeutic agent comprising stem cells isolated from the chimeric mesenchymal stem cell group or cells differentiated therefrom as an active ingredient.
  • Another object of the present invention is to provide a method for differentiating parathyroid tissue cells from tonsil stem cells.
  • Still another object of the present invention is to provide a method for producing parathyroid hormone using tonsil stem cells.
  • Still another object of the present invention is to provide parathyroid tissue cells differentiated from tonsil stem cells.
  • Still another object of the present invention is to provide a method for treating parathyroid dysfunction or osteoporosis, comprising administering a cell therapy comprising parathyroid tissue cells to a subject suspected of hypothyroidism or osteoporosis.
  • the chimeric mesenchymal stem cell group of the present invention can be produced not only from the tonsil tissue discarded, but also from a separate tonsil tissue, and thus can be produced economically, and since it shows the same stem cell activity even after freezing and thawing, stem cells It can be used as a cell source for banks.
  • the method of differentiating parathyroid tissue cells from tonsill stem cells of the present invention can effectively differentiate parathyroid tissue cells in a short time by using tonsil tissue discarded after tonsillectomy, the parathyroid tissue cells obtained therefrom are present in the body It can produce parathyroid hormone in the same way as parathyroid tissue cells and regulate the secretion of parathyroid hormone according to the calcium concentration outside the cell, so it can be effectively used for the treatment of diseases requiring parathyroid hormone including hypothyroidism and osteoporosis.
  • a large amount of parathyroid hormone is secreted out of the cells so that parathyroid hormone can be easily obtained.
  • T-MSCs express specific surface antigens.
  • T-MSCs can differentiate into mesodermal and endoderm cells.
  • Figure 5 is a diagram illustrating the process of separating the amygdala stem cells from tonsils obtained by tonsillectomy.
  • FIG. 6 is a view showing the change in cell shape according to differentiation period from tonsil derived cells.
  • FIG. 7 is a view showing the results of the PTH concentration irradiation secreted into the cell culture medium during the differentiation period.
  • FIG. 8 is a view showing the expression pattern of PTH protein according to the parathyroid differentiation period of tonsil cells.
  • Figure 9 shows the distribution of PTH in the intracellular matrix during differentiation into parathyroid tissue.
  • FIG. 10 is a diagram showing the expression and distribution of CHGA, a PTH secretion related protein.
  • 11 is a diagram showing the expression distribution of cells of PTH and CHGA.
  • FIG. 12 is a diagram showing the regulation of PTH secretion according to the extracellular calcium concentration change.
  • FIG. 13 is a view showing the change in the cell shape of tonsil stem cells according to the extracellular calcium concentration change.
  • FIG. 14 is a view showing the change in the expression of calcium-sensitive receptors in tonsil stem cells according to the extracellular calcium concentration changes.
  • 15 is a diagram showing the cell microstructure of tonsil stem cells differentiated into parathyroid tissue.
  • 16 is a view showing the bone formation capacity of PTH produced and secreted in tonsil stem cells.
  • the present invention is a method for producing a chimeric mesenchymal stem cell population comprising the step of mixed culture of tonsil-derived mesenchymal stem cells obtained from a separate donor Or it provides a method for mixed culture of tonsil-derived mesenchymal stem cells.
  • the method for producing a chimeric mesenchymal stem cell group of the present invention or a method for mixed culture of mesenchymal stem-derived mesenchymal stem cells comprises the steps of: (i) obtaining mononuclear cells from each tonsil tissue isolated from a separate donor; (ii) culturing the obtained mononuclear cells to select mesenchymal stem cells; And, (iii) mixing and culturing the selected mesenchymal stem cells.
  • the term "tonsil” or “tonsil” is located at the back of the neck and the back of the nose, primarily to protect the body from substances such as bacteria that invade from the outside and act as lymphatic epithelial immune tissue. Means the organization to perform;
  • the amygdala includes a pharyngeal tonsil, ear pharyngeal tonsil, palate tonsil, tongue tonsil, and the like.
  • the amygdala is used as a source tissue for providing tonsil-derived mesenchymal stem cells.
  • mesenchymal stem cells refers to undifferentiated stem cells that can be differentiated into bone, cartilage, fat, myeloid epilepsy, muscle, nerves, and the like. But also in cord blood, peripheral blood, and other tissues.
  • the mesenchymal stem cells are not particularly limited thereto, but may be cells that can be mixed and cultured with each mesenchymal stem cell derived from a person's tonsil and derived from different individuals, and are adipocytes, bone Not only can they differentiate into mesodermal tissue cells such as cells, chondrocytes, but also endoderm tissue cells such as parathyroid tissue cells.
  • stem cell refers to a cell having self-replicating ability and capable of differentiating into two or more new cells, and includes totipotent stem cells and pluripotent stem cells. And multipotent (pluripotent) stem cells.
  • Tototent stem cells are pluripotent cells that can develop into a complete individual. Cells up to 8-cells after fertilization of eggs and sperm have this property and isolate the cells. When transplanted into the uterus means a cell that can develop into a complete individual.
  • Pluripotent stem cells are cells that can develop into various cells and tissues derived from ectoderm, mesoderm, and endodermal layer, and are located inside the blastocyst appearing 4-5 days after fertilization. It is derived from the inner cell mass, which is called embryonic stem cells, and refers to cells that differentiate into various other tissue cells but do not form new life.
  • fat cell refers to a major cell constituting adipose tissue that stores energy in the form of fat.
  • the adipocytes may exist in two forms: white fat cells comprising large fat droplets surrounded by a cytoplasmic layer and polygonal brown fat cells comprising a significant amount of cytoplasm with evenly dispersed fat droplets.
  • chondrocytes refers to the only cells found in the cartilage, and can produce and maintain a cartilage substrate mainly composed of collagen and proteoglycans.
  • the composition of chondrocytes in cartilage depends on the type of cartilage and the location of the tissue.
  • bone cell refers to a cell that is differentiated from osteoblasts in developing bone, which is present inside the bone cavity contained in bone tissue, has a flat ellipsoidal form of the same type as the bone cavity, and is extremely It has many processes and is involved in mineral metabolism of surrounding bone tissue.
  • parathyroid tissue cells refers to cells constituting the parathyroid gland, an endocrine organ attached to the thyroid gland, secretes parathormone and regulates metabolism of calcium and phosphorus in body fluids. In the present invention, as long as the parathyroid hormone is secreted, it may be included in parathyroid tissue cells.
  • a method for obtaining mononuclear cells from tonsil tissue is not particularly limited thereto, and may be obtained by grinding the tonsil tissue and filtering the same. .
  • the method for selecting mesenchymal stem cells by culturing mononuclear cells is not particularly limited thereto, and may be performed by selecting cells attached to the bottom of the culture vessel by culturing using a stem cell culture medium.
  • the stem cell culture medium is not particularly limited to this, but DMEM-HG (Dulbecco's modified Eagle's medium-High Glucose) medium, including FBS (Invtrogen) and antibiotics, Keratinocyte-SFM, RPMI-1640 may be used,
  • the antibiotic is not particularly limited thereto, but may be streptomycin, ampicillin, or the like.
  • mixed culture refers to a method of mixing and culturing mesenchymal stem cells isolated from tonsils obtained from different separate donors. Even in the case of tissue cells of the same character, cells separated from different donors have different characteristics of the cells, and thus do not exhibit uniform characteristics. However, tonsil-derived mesenchymal stem cells of the present invention exhibit the same characteristics as stem cells obtained from a single donor even when cultured by mixing stem cells obtained from different donors.
  • the present invention provides a chimeric mesenchymal stem cell population prepared by the above method.
  • Said chimeric mesenchymal stem cell population comprises (i) mesenchymal stem cells isolated from separate tonsil tissue obtained from separate donors; (ii) exhibit the same cellular activity as tonsil-derived mesenchymal stem cells obtained from a single donor; (iii) the expression of CD14, CD34 and CD45 is reduced; (iv) the expression of CD73, CD95 and CD105 is increased; (v) negative for immunorelevant expressing antibodies; (vi) can differentiate into mesodermal cells; And (vii) differentiate into endoderm cells.
  • chimeric mesenchymal stem cell group refers to mesenchymal stem cells obtained by mixed culture of tonsil-derived mesenchymal stem cells obtained from separate donors, wherein the chimeric mesenchymal stem cell groups are different donors. Each mesenchymal stem cell isolated from is included, but exhibits the same level of cellular characteristics as the mesenchymal stem cells isolated from a single donor.
  • the chimeric mesenchymal stem cell population may exhibit the phenotype of a typical stem cell immunophenotype of T-MSC. That is, the expression of CD14, CD34 and CD45 is weak while the expression of CD73, CD95 and CD105 is increased (FIG.
  • FIG. 2A the immune-related expressing antibodies
  • HLA-DR the immune-related expressing antibodies
  • CD40, CD80, CD86 the immune-related expressing antibodies
  • FIG. 2B the immune-related expressing antibodies
  • they can be differentiated into mesodermal cells, adipocytes, bone cells and chondrocytes (FIG. 3A), and also into endoderm cells, parathyroid tissue cells (FIG. 3B).
  • chimera when mixed cultured tonsil-derived mesenchymal stem cells obtained from separate donors, chimera was formed to form one mesenchymal stem cell (Fig. 4b). ), Mesenchymal stem cells obtained from a mixed culture of tonsil-derived mesenchymal stem cells obtained from a single donor and tonsil-derived mesenchymal stem cells obtained from a separate donor were compared in terms of CFU-F formation ability and proliferative capacity. The results confirmed similar levels (FIG. 1A).
  • the present invention provides a method for producing a chimeric mesenchymal stem cell group having improved proliferative capacity, including freezing and thawing the chimeric mesenchymal stem cell group.
  • the chimeric mesenchymal stem cell group having improved proliferative capacity may exhibit (i) differentiation capacity equivalent to that of the chimeric mesenchymal stem cell group that has not undergone freezing and thawing; And, (ii) an improved level of proliferative capacity than the chimeric mesenchymal stem cell population that did not perform freezing and thawing.
  • freeze of the present invention refers to the action of freezing using a cooling medium such as liquid nitrogen in order to preserve the tonsil-derived mesenchymal stem cells of the present invention.
  • a solution containing a cryoprotectant may be used to prevent cell damage during freezing.
  • freeze protection agent refers to a substance which is added to a liquid medium for the purpose of alleviating the East Sea when biological cells are stored alive in a frozen state, and the freeze protection agent is not particularly limited thereto. However, glycerol, sugar, glucose and the like can be used.
  • thawing in order to utilize the frozen tonsil-derived mesenchymal stem cells of the present invention, means the action to increase the temperature to room temperature so that the cells exhibit normal physiological activity.
  • enhanced proliferative capacity of the present invention refers to a phenomenon in which the rate of proliferation of tonsil-derived mesenchymal stem cells provided by the present invention increases after applying a freezing and thawing process.
  • the mesenchymal stem cells obtained by mixing and cultured tonsil-derived mesenchymal stem cells obtained from a single donor and tonsil-derived mesenchymal stem cells obtained from a separate donor are frozen and thawed.
  • Mixed cultured mesenchymal stem cells were confirmed to exhibit a relatively high level of proliferative capacity (FIG. 1B).
  • the tonsil-derived mesenchymal stem cells of the present invention can be differentiated into mesodermal cells and endoderm cells, and show the same differentiation and proliferative capacity as stem cells derived from a single donor even when mixed with other donor stem cells. In addition, even after freezing and thawing, it is possible to maintain the differentiation capacity and proliferative capacity of the first stem cells as they are, and thus can be utilized as a cell source essential for forming a stem cell bank.
  • the present invention provides a method for differentiating the chimeric mesenchymal stem cell population into various cells.
  • the method of differentiating the chimeric mesenchymal stem cell group into various cells is not particularly limited, but the chimeric mesenchymal stem cell group is differentiated into chondrocytes containing ascorbic acid, L-proline, TGF-3, BMP-6 and ITS.
  • chimeric mesenchymal stem cell group was cultured in culture medium containing ascorbic acid 2-phosphate, dexamethasone and ⁇ -glycerophosphate to differentiate into bone cells, or the chimeric mesenchyme Stem cell populations are cultured in culture medium containing 3-isobutyl-1-methyl-gentine, dexamethasone, indomethacin and insulin to differentiate into adipocytes, or the chimeric mesenchymal stem cell population is activated by activin A (activin A). ) And Shh (sonic hedgehog) can be cultured in a culture medium containing the parathyroid tissue cells.
  • the term "differentiation" of the present invention generally refers to a phenomenon in which a relatively simple system is separated into two or more qualitatively different sub systems, specifically, during cell division and growth. It refers to a phenomenon in which structures and functions are specialized to each other, that is, a phenomenon in which a form or a function of a cell or tissue of an organism changes to perform a task given to each. For example, qualitatively between parts of a biological system that were initially nearly homogeneous, such as head or torso distinctions between eggs that were initially homogenous in population development, or cells such as myocytes or neurons. Phosphorus difference arises, or as a result, the phenomenon divided into sub division or sub-system which can be distinguished qualitatively is mentioned.
  • the method for measuring the degree of differentiation of the chimeric mesenchymal stem cell group differentiated by the above method is not particularly limited to this, using flow cytometry, immunocytochemical method, PCR and gene expression profile which are well known in the art.
  • -PCR Oil red O staining, Safranin O staining, Type II collagen immunohistochemical staining, alkaline phosphate staining and Alizarin S staining can be used.
  • tonsil mesenchymal stem cells (tonsillar mesenchymal stem cells, T-MSC) was confirmed that can be differentiated into adipocytes, bone cells and chondrocytes that are mesodermal cells (Fig. 3a) ), As well as parathyroid tissue cells that are endoderm cells can be differentiated (Fig. 3b).
  • the present invention provides a cell therapeutic agent comprising stem cells isolated from the chimeric mesenchymal stem cell group, or cells differentiated from the chimeric mesenchymal stem cell group as an active ingredient.
  • the differentiated cells are not particularly limited thereto, but may preferably be all cells that can be used for cell therapy, more preferably may be mesodermal cells or endoderm cells, most preferably fat Cells, chondrocytes, osteocytes, parathyroid tissue cells, and the like.
  • cellular therapeutic agent refers to 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 manipulation from an individual.
  • drugs used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and screening live autologous, allogeneic, or heterologous cells in vitro or by altering the biological characteristics of the cells in order to restore tissue function. Means.
  • the chimeric mesenchymal stem cell population of the present invention is a variety of therapeutic protocols in which the tissues or organs of the body are fortified, treated or replaced by engraftment, transplantation or infusion of a desired cell population, for example, a stem cell or a derived cell population.
  • a desired cell population for example, a stem cell or a derived cell population.
  • the chimeric mesenchymal stem cell population of the present invention can replace or enhance existing tissue, resulting in new or changed tissue, or bind to biological tissue or structure.
  • the chimeric mesenchymal stem cell group of the present invention can be used for strengthening, treating or replacing human cartilage, tendons, ligaments, parathyroid tissues and the like.
  • the invention is a parathyroid tissue cell from tonsils, comprising culturing tonsils in a medium comprising Activin A and Sonic hedgehog (Shh) It provides a way to differentiate.
  • parathyroid tissue when inducing parathyroid tissue from embryonic stem cells or adult stem cells derived from the thymus, it took longer than 6 months to induce parathyroid tissue.
  • the present invention first identified a method capable of inducing differentiation of parathyroid tissue in a short period of time from amygdala stem cells, which have never been considered as candidates for differentiation into parathyroid tissue.
  • the terms "tonsil”, “stem cell”, “parathyroid tissue cells” are as described above, in the present invention, the tonsil is used as a source tissue for providing tonsil-derived mesenchymal stem cells.
  • tonsils means mesenchymal stem cells (T-MSCs) derived from tonsils.
  • Parathyroid tissue cell differentiation method of the present invention comprises culturing the amygdala stem cells in a medium containing activin A and sonic hedgehog (Shh) at a concentration of 50 ng / ml ⁇ 300 ng / ml, respectively,
  • the culture period of the cells is preferably 4 days or more, more preferably 7 days or more.
  • the amygdala stem cells may be collected from tonsil tissue isolated from a patient who has undergone tonsillectomy, but are not limited thereto.
  • the collected tonsil is washed with buffer, and then treated with enzyme mixture to decompose the connective tissue.
  • the suspended tissue is centrifuged and the cell mixture in the cut tissue is ficol Immunohistochemical method using cell surface markers among the cells showing adhesion to the bottom of the culture plate after culturing only the precipitated part of the lower layer by cell fractionation and centrifugation using Tonsillar stem cells can be isolated using
  • activin A which is used as a differentiation inducing agent, is a member of the TGF-beta superfamily, first known as a protein that promotes FSH release, but it is known as mesoderm induction, neuronal differentiation, bone remodeling, hematopoiesis, It is known to have a wide range of biological activities.
  • Sonic hedgehog proteins are one of three proteins in a family of mammalian signaling pathways called hedgehogs, which play a key role in regulating vertebrate organogenesis such as finger growth in the extremities and brain organization.
  • Sonic hedgehog is a type of morphogen that has different effects on developing embryonic cells depending on its concentration, and is known to control cell division of adult stem cells and to induce some cancers. .
  • activin A and sonic hedgehog can be purchased by using any commercially available activin A and sonic hedgehog.
  • the concentrations of activin A and sonic hedgehog are less than 50 ng / ml, the period required for differentiation of parathyroid tissue cells from amygdala stem cells may be extended to several months or the differentiation efficiency may be inferior. If the concentration of the differentiation induction agent exceeds 300 ng / ml, there is no improvement in the efficiency of differentiation induction, it is inefficient to use such a high concentration of chemicals.
  • the concentrations of Activin A and Sonic Hedgehog are each 80 ng / ml to 200 ng / ml, most preferably 100 ng / ml each.
  • the parathyroid tissue from the amygdala stem cells may be shortened only by 4 days of culture. Can differentiate cells.
  • the method of the present invention to induce differentiation of parathyroid tissue cells by culturing tonsil stem cells in a medium containing activin A and sonic hedgehog at a concentration of 100 ng / ml, respectively, for 4 to 25 days. If the culture period exceeds 4 days, the medium can be replaced with fresh medium containing activin A and sonic hedgehog every 4-5 days.
  • the medium used to induce differentiation may be used a conventional medium that can be used for the culture of stem cells, for example, DMEM (Dulbecco's modified Eagle medium), Keratinocyte-SFM (Keratinocyte serum free medium), RPMI-1640, and the like, and preferably, DMEM medium may be used.
  • the stem cell medium may be supplemented with an additive.
  • an additive may contain neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions.
  • neutral buffers such as phosphates and / or high concentrations of bicarbonate
  • protein nutrients such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine
  • lipids fatty acids, cholesterol, HDL or LDL extracts of serum
  • other components found in most preservative media of this kind such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt
  • Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as ⁇ -mercaptoethanol.
  • the parathyroid hormone produced by parathyroid tissue cells after 4 days of culture as a result of incubating the amygdala stem cells in a medium containing activin A and sonic hedgehog at a concentration of 100 ng / ml, respectively.
  • the production of was significantly increased, and the intracellular microstructure was confirmed using electron microscopy.
  • the differentiated cells were differentiated into hormone secretion tissues because of the presence of a basic granular microstructure and a granular and granular precursor structure. could be confirmed.
  • the present invention provides a method for producing parathyroid hormone using tonsilocytes.
  • the present invention comprises the steps of i) culturing amygdala stem cells in a medium containing Activin A and Sonic hedgehog (Shh) to differentiate into parathyroid tissue cells, and ii) i) step It provides a method for producing parathyroid hormone using tonsils, comprising the step of separating the parathyroid hormone from the culture solution or cell lysate obtained in the.
  • parathyroid tissue cells differentiated from tonsil stem cells produce and secrete parathyroid hormone like normal parathyroid tissue cells. Therefore, parathyroid hormone can be obtained by separating parathyroid hormone from culture or cell lysate of parathyroid tissue cells differentiated from tonsil stem cells.
  • tonsil stem cells, activin A, and sonic hedgehog are as described above.
  • the step of culturing the amygdala stem cells in a medium containing activin A and sonic hedgehog to differentiate into parathyroid tissue cells is the same as described above.
  • Separation of parathyroid hormone from the culture or cell lysate may be carried out using a general method that can be used for the separation and purification of proteins, for example, affinity chromatography, ion exchange chromatography, coprecipitation, etc.
  • the method may be used, but the present invention is not limited thereto and may be appropriately selected by a person skilled in the art.
  • parathyroid hormone is produced in the parathyroid tissue cells and then present in the cells in the form of secretory granules, depending on the extracellular calcium concentration is determined whether the secretion outside the cell.
  • the method of the present invention in order to facilitate the secretion of parathyroid hormone produced by parathyroid tissue cells derived from tonsil stem cells to facilitate the separation of parathyroid hormone, the method of the present invention is the step i) Thereafter, adjusting the calcium concentration of the culture medium may further include secreting parathyroid hormone out of the cells.
  • the method may further comprise adjusting the calcium concentration of the culture medium to less than 1.5 mM.
  • the calcium concentration of the culture medium for promoting secretion of parathyroid hormone is preferably less than 1.0 mM, more preferably less than 0.5 mM, and most preferably less than 0.1 mM.
  • the parathyroid hormone may be lysed to separate the parathyroid hormone from the lysate without secreting the parathyroid hormone out of the cell.
  • Parathyroid hormone production method of the present invention by using tonsil stem cells that can be obtained from tonsil tissue discarded after tonsillectomy, to facilitate the production of parathyroid hormone that can be useful for the treatment of parathyroid hypoplasia or osteoporosis, etc.
  • tonsil stem cells that can be obtained from tonsil tissue discarded after tonsillectomy, to facilitate the production of parathyroid hormone that can be useful for the treatment of parathyroid hypoplasia or osteoporosis, etc.
  • it is possible to easily obtain a large amount of hormones by promoting the secretion of parathyroid hormone through the regulation of calcium concentration outside the cell.
  • the amygdala stem cells are cultured in a medium containing activin A and sonic hedgehog to induce differentiation of parathyroid tissue and confirm the expression patterns of parathyroid hormone in each period. It was confirmed that the expression of parathyroid hormone was significantly increased from the point of time elapsed, and that the expression level was significantly increased after 7 days after induction of differentiation, thereby making the parathyroid tissue differentiated from tonsil stem cells by the method of the present invention. It was confirmed that parathyroid hormone could be produced from the cells.
  • the cumulative secretion of parathyroid hormone increased as the differentiation-inducing period elapsed, and it was confirmed that a large amount of parathyroid hormone can be obtained by continuously culturing parathyroid tissue differentiated from tonsil stem cells.
  • the external calcium concentration is lower than the normal concentration (1.5 mM)
  • it is confirmed that the parathyroid hormone is secreted to the outside of the cell it is also possible to control the secretion of parathyroid hormone by controlling the calcium concentration of the culture medium when inducing differentiation of parathyroid tissue cells It was.
  • the present invention provides parathyroid tissue cells differentiated from tonsil stem cells.
  • parathyroid tissue cells of the present invention can be obtained by the parathyroid tissue cell differentiation method comprising culturing tonsils in the medium containing the amygdala stem cells as described above, activin A and Sonic hedgehog, preferably
  • the amygdala stem cells can be obtained by culturing at least 4 days in a medium containing activin A and sonic hedgehog at a concentration of 50 ng / ml to 300 ng / ml, respectively, which is most preferred for obtaining parathyroid tissue cells.
  • the concentrations of activin A and sonic hedgehog are 100 ng / ml each.
  • Parathyroid tissue cells obtained by differentiation from amygdala stem cells by the differentiation method of the present invention as described above can produce parathyroid hormone, and can control the secretion of parathyroid hormone in response to changes in calcium concentration outside the cell, If the calcium concentration is lower than the normal concentration (1.5 mM) to secrete parathyroid hormone outside the cell, if the calcium concentration outside the cell is higher than the normal concentration can suppress the secretion of parathyroid hormone.
  • the parathyroid tissue cells of the present invention can function as parathyroid tissue cells, like parathyroid tissue cells present in the body, and are treatable by diseases or parathyroid hormone due to impaired function of parathyroid tissue or lack of parathyroid hormone. It can be usefully used for the treatment of known osteoporosis and the like.
  • the present invention provides a cell therapy agent of hypothyroidism or osteoporosis comprising the parathyroid tissue cells of the present invention.
  • cell therapeutic agent is as described above.
  • the cell therapy agent comprising the parathyroid tissue cells of the present invention can be used include, for example, hypothyroidism or osteoporosis.
  • hypothyroidism is a disease caused by hypocalcemia caused by lowered calcium in the blood due to parathyroid hormone dysfunction.
  • the only way to treat it is to take excess calcium, but to take excess calcium instead of parathyroid hormone.
  • osteoporosis refers to a condition in which bone fractures occur due to a decrease in bone quantity due to a decrease in the amount of bone and qualitative changes
  • an agent that mainly suppresses bone resorption is mainly used.
  • the use of parathyroid hormone has been suggested as a direct stimulator of bone formation.
  • the use of parathyroid hormone can only be used for a short period of time (up to two years, US FDA), expensive, subcutaneous injections such as daily insulin injections, and rapid bone loss after the end of treatment. It has been reported. Therefore, if you use a cell therapy containing parathyroid tissue that can produce parathyroid hormone rather than administration of parathyroid hormone and can regulate hormone secretion according to the extracellular calcium concentration, osteoporosis can be treated without side effects. .
  • treatment in the present invention means any action that improves or benefits the condition of the disease by administration of the cell therapy.
  • the cell therapy agent of the present invention contains 1.0 ⁇ 10 5 to 1.0 ⁇ 10 9 cells, preferably 1.0 ⁇ 10 6 to 1.0 ⁇ 10 8 cells, more preferably 1.0 ⁇ 10 7 cells per ml. can do.
  • the cell therapy of the invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) can be added to the cell population prior to freezing.
  • standard cryopreservatives eg DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)
  • the cell therapy agent may be administered by formulating a unit dosage form pharmaceutical preparation suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, wherein the preparation is effectively administered by one or several administrations. Contains the amount.
  • Formulations suitable for this purpose include parenteral injectables such as injectable ampoules, injectables such as infusion bags, sprays such as aerosol preparations and the like.
  • the injection ampoule may be mixed with the injection solution immediately before use, and physiological saline, glucose, mannitol, and Ringer's solution may be used as the injection solution.
  • the infusion bag can also be made of polyvinyl chloride or polyethylene and can be used in Baxter, Becton Dickinson, Medcep, National Hospital Products or Terumo.
  • the injection bag of the yarn can be illustrated.
  • one or more pharmaceutically acceptable conventional inert carriers such as preservatives, analgesic agents, solubilizers or stabilizers in the case of injections, etc. It may further include a base, excipients, lubricants or preservatives.
  • the cell therapy or pharmaceutical preparation of the present invention thus prepared may be administered with other stem cells used for transplantation and other uses, or in the form of mixtures with such stem cells, using administration methods commonly used in the art.
  • administration methods commonly used in the art.
  • the administration can be both non-surgical administration using a catheter and surgical administration methods such as injection or transplantation after dissection of the disease site, but non-surgical administration using a catheter is more preferable.
  • parenteral administration for example, in addition to direct lesions, transplantation by intravascular injection, which is a general method of hematopoietic stem cell transplantation, is also possible according to a conventional method.
  • the daily dose of the stem cells may be administered 1.0 ⁇ 10 4 to 1.0 ⁇ 10 10 cells / kg body weight, preferably 1.0 ⁇ 10 5 to 1.0 ⁇ 10 9 cells / kg body weight divided into one or several times .
  • the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore, the dosage may be It is not intended to limit the scope of the present invention in terms of aspects.
  • the present invention provides a method for treating parathyroid function-related diseases or osteoporosis, comprising administering a cell therapy comprising parathyroid tissue cells to an individual suspected of hypothyroidism or osteoporosis. Provide a method.
  • Administration of the cell therapy of the present invention is applicable to any animal, and animals include humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, mice, rats, and cats.
  • the term "administration" means introducing the cell therapy of the invention into a patient in any suitable manner and includes the transplantation of differentiated cells. Routes of administration of the cell therapy products of the invention can be administered via a variety of routes as long as they can reach the desired tissue.
  • Example 1 Obtaining Amygdala Mesenchymal Stem Cells
  • 5 is a method for isolating and culturing stem cells from the obtained tonsil tissue.
  • the collected tonsils were washed with physiological saline, and the enzyme mixture 210 U / mL collagenase type I (Invitrogen) and 10 ⁇ g / mL DNAse (Sigma-Aldrich, St. Louis, MO). ) was mixed in the culture medium RPMI-1640 (Roswell Park Memorial Institute medium 1640, Invitrogen Corporation, Carlsbad, Calif.) For 30 minutes at 37 °C conditions connective tissue in the amygdala. The suspended tissue was centrifuged and filtered to obtain cells. The obtained cells were washed twice with RPMI 1640/20% NHS (normal human serum) (PAA Laboratories, GmbH, Paching, Austria) and once with RPMI 1640/10% NHS.
  • RPMI-1640 normal human serum
  • Washed cells were subjected to Ficoll-Paque (GE Healthcare, Little Chalfont, Buckinghamshire, UK) gradient centrifugation to obtain mononuclear cells.
  • Dulbecco's modified Eagle's medium-High Glucose (DMEM-HG) containing 10 8 fetal mononuclear cells (Fetal bovine serum, FBS, Invitrogen), 100 ⁇ g / ml streptomycin and 100 U / ml ampicillin (Invitrogen) The medium was inoculated and incubated for 48 hours. After incubation, unattached cells are removed, and adherent cells, considered tonsillar mesenchymal stem cells (T-MSCs), are passaged to 3-5 generations over 4 weeks. Used.
  • T-MSCs tonsillar mesenchymal stem cells
  • T-MSCs incubated above were washed with PBS (pH 7.4), and suspended by adding cryodium (50% FBS, 40% DMEM and 10% DMSO) to the washed T-MSCs. Aliquots were included to contain 2 ⁇ 10 6 cells per vessel.
  • the suspension dispensed vessel was frozen at ⁇ 70 ° C. for 24 hours and stored in ⁇ 200 ° C. liquid nitrogen for 10 days (Liu Y, et al., Biotechnol. Prog., 26: 1635-1643, 2010).
  • the stored vessel was thawed in 37 ° C. water for 2 minutes using a known method, and the thawed cell suspension was centrifuged to obtain T-MSC.
  • cryopreserved-and-thawed cells which have undergone freezing and thawing, were cultured for 7 days and applied to the same method as described above.
  • FC cultures obtained from a single donor there was no difference between FC cultures obtained from a single donor and mixed FC cultures obtained from several donors in terms of proliferative capacity (FIG. 1B).
  • mixed CTC cultures obtained from several donors showed relatively high levels of proliferative capacity.
  • T-MSC Cell phenotype of T-MSC was analyzed by using FACSCalibur system (Becton Dickinson, Franklin Lakes, NJ) to confirm the expression of the surface antigens of the FC and CTC.
  • FC and CTC were incubated for 14 days and 7 days, respectively, and stained at 4 ° C. for 30 minutes using respective antibodies (2 ⁇ g / ml) capable of reacting with surface antigen and analyzed using the FACSCaliur system. .
  • the antibodies used are as follows; Hematopoietic cell markers (PE-CD14, PE-CD34 and PerCP-CD45), primitive cell markers (PE-CD73, PE-CD90, and PE-CD105), class II MHCs and auxiliaries Co-stimulatory molecules markers (PE-HLA-DR, FITC-CD40, PE-CD80 and FITC-CD86) and follicular dendritic cell markers (PE-CD11b, PE-CD21, PE -CD23, PE-CD35 and PE-CD54).
  • Hematopoietic cell markers PE-CD14, PE-CD34 and PerCP-CD45
  • primitive cell markers PE-CD73, PE-CD90, and PE-CD105
  • Co-stimulatory molecules markers PE-HLA-DR, FITC-CD40, PE-CD80 and FITC-CD86
  • follicular dendritic cell markers PE-CD11b, PE-CD21, PE -CD23,
  • an IgG1 or IgG2a (Becton Dickinson) isotype antibody, which is an antibody capable of performing nonspecific binding, was used as a control.
  • the immunophenotype of T-MSC showed a typical stem cell phenotype. That is, the expression of CD14, CD34 and CD45 was weak while the expression of CD73, CD95, CD105 was increased, indicating that T-MSC was different from hematopoietic stem cells (FIG. 2A). In addition, immune-related expressing antibodies (HLA-DR, CD40, CD80, CD86) were also negative (FIG. 2B).
  • T-MSC is a different cell from FDC.
  • T-MSCs were used to induce differentiation into mesodermal cells, adipocytes, bone cells and chondrocytes.
  • adipocyte culture Invtorgen
  • T-MSCs were inoculated into a 14 ml conical tube containing chondrocyte culture medium (Invtorgen) containing dexamethasone, ascorbic acid, L-proline, TGF-3, BMP-6 and ITS. Incubate for 3 weeks with replacement every 3-4 days. After the incubation was terminated, the cultured cells were harvested, and the harvested cells were fixed with 4% paraformaldehyde and embedded with paraffin to obtain paraffin blocks. The obtained paraffin block was cut to a thickness of 5 ⁇ m to obtain flakes. The obtained flakes were placed on a slide glass, and then paraffin was removed to fix cell sections on the slide glass.
  • chondrocyte culture medium Invtorgen
  • T-MSCs were induced to differentiate into parathyroid cell-like cells, which are endoderm cells.
  • T-MSC RPMI 1640 medium containing 5% FBS, activin A (activin A, 100 ng / ml, R & D System, Inc. Minneapolis) and sonic hedgehog (Shh) (100 ng / ml, R & D Systems) Inoculated and cultured for 3 weeks while replacing the culture every 4 days. After the incubation was completed, the expression of endoderm-specific protein was confirmed by reverse transcriptase chain reaction (RT-PCR) method.
  • RT-PCR reverse transcriptase chain reaction
  • GCM2 glial cells missing homolog 2
  • CaSR calcium sensing receptor
  • CCL21 specific for parathyroid tissue cells chemokine (CC motif) ligand 21
  • PTH parathyroid hormone
  • RT system Promega, Madison, Wis.
  • the sequence of the primer used is described in Table 1, the overall method was performed according to the user's manual of TRIZOL (Invitrogen).
  • FoxA2 forkhead box A2, SOX17: sex determining region Y (SRY) -box 17, CXCR4: chemokine (CXC motif) receptor 4, Eya1: eyes absent homolog 1, Six1: SIX homeobox 1, Pax1: paired box 1, BMP4: bone morphogenic protein 4, GCM2: glial cells missing homolog 2, CaSR: calcium sensing receptor, CCL21: chemokine (CC motif) ligand 21, PTH: parathyroid hormone, and GAPDH: glyceraldehyde-3-phosphate dehydrogenase
  • T-MSCs can be differentiated into parathyroid tissue cells, which are endoderm cells.
  • Human CD4 + T cells were obtained from the peripheral blood of healthy donors and PMA (Phorbol 12- myristate 13-acetate) (Sigma-Aldrich) was used as a mitosis promoter.
  • CD4 + T cells were labeled with carboxyfluorescein diacetate (succinimidyl ester) (CFSE).
  • CFSE carboxyfluorescein diacetate
  • the specific method is as follows: The cells were suspended in PBS containing 0.1% BSA pre-warmed at a concentration of 10 6 cells / ml, followed by the addition of 10 ⁇ M CFSE. Subsequently, the cells were incubated at 37 ° C. for 10 minutes, and the cells were left in 5 times ice-cold culture to terminate staining, followed by centrifugation to obtain CD4 + T cells labeled with CFSE.
  • the obtained CFSE-labeled CD4 + T cells (3 ⁇ 10 4 cells) were resuspended in culture and washed three times, and the washed cells were wells (10 4 cells) with or without cultured T-MSCs or not. The wells were inoculated. Then, PMA at a final concentration of 1 to 200 ng / ml was added to each well inoculated with the cells, and cultured for 3 days, and then the proliferation of CFSE + CD + T cells was measured by flow cytometry, and ModiFitLT The reduction of CFSE positive cells was analyzed using the software.
  • Each T-MSC from three independent donors was incubated for four weeks, either individually or mixed together. After the incubation was completed, chimerism was confirmed by extracting genomic DNA from each cultured cell and performing STR analysis by a known method (Kristt DM, et al., J. Biomol. Tech., 16: 380-391, 2005; Kim DW, et al., Blood, 103: 1941-1948, 2004). At this time, the extracted genomic DNA was amplified by PCR, STR analysis was performed using capillary electrophoresis (ABI 3130Xl Genetic analyzer, Carlsbad, CA), and the result analysis was performed using GeneMapper software.
  • chimeras (heterozygote) were formed at three sites of D8S1179, D3S1358 and D18S51 among the total 15 sites (FIG. 4B).
  • T-MSCs derived from amygdala of various origins can form chimeras in mixed cultures, and thus, T-MSCs exhibited similar and similar levels of cellular characteristics regardless of origin, resulting in mixed Through culture, it was found that one mesenchymal stem cell group could be formed.
  • T-MSCs For further study of the differentiation of amygdala stem cells into parathyroid tissue, differentiation of T-MSCs from single individuals into parathyroid cell-like cells was induced. Specifically, 5% of T-MSCs were induced. Inoculate DMEM medium containing FBS, activin A (activin A, 100 ng / ml, R & D System, Inc. Minneapolis) and sonic hedgehog (Shh, 100 ng / ml, R & D Systems), and incubate the culture every 4 days Incubate for 21-25 days with replacement.
  • activin A activin A
  • sonic hedgehog Shh, 100 ng / ml, R & D Systems
  • amygdala stem cells formed a multi-layer accumulating into several layers as shown in FIG.
  • PTH Whenever exchanged with cell culture medium containing differentiation inducer by differentiation period (0, 4, 7, 10, 15, 20 days), PTH is collected and secreted into the extracellular cells during differentiation into parathyroid tissue. It was used as a sample for analyzing the concentration of.
  • Cell cultures collected at each time were filtered through a 0.45 ⁇ m syringe filter to remove cell suspensions and cell debris, and the filtered cell cultures were freeze-dried and concentrated using a freeze dryer (Operon).
  • Lyophilized powder samples were resuspended in phosphate buffered saline (pH 7.4) and analyzed for PTH concentration by immunochemistry. That is, the concentration of PTH released by the ELCIA method using the binding force with the antibody to the PTH protein was investigated.
  • the cells treated with the differentiation inducing liquid secreted statistically significant amount of PTH into the cell culture fluid compared with the cells that did not differentiate.
  • the increase was significantly increased from 7 days to 10 days after the differentiation induction solution, and the amount was up to 40 pg / ml, which was 15-60 pg / ml of normal PTH concentration range in vivo. It was found to be included in the range (FIG. 7B).
  • T-MSCs were collected during differentiation, the cell surface was washed twice with PBS (Phosphate buffered saline, pH 7.4), and then RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP- 40, 1% sodium deoxycholate, 0.1% SDS) was used to extract the intracellular protein portion.
  • the extracted protein was quantified by BCA (bicinchoninic acid) method using albumin as a standard solution, and used for intracellular PTH protein expression.
  • SDS-PAGE 10-12% SDS (sodium deoxycholate) -polyacrylamide gel (SDS-PAGE) was prepared, and 30 ⁇ g of protein samples quantified at each stage of differentiation period were loaded on pre-prepared SDS-PAGE, followed by electrophoresis. My proteins were separated by their size. After electrophoresis, proteins of each size separated on the gel were transferred to the nitrocellulose membrane using an electric method.
  • the primary antibody-specific secondary antibody mouse-IgG-HRP (horse radish peroxidase) conjugate was further reacted (diluted at 1: 3000 ratio).
  • the response of the PTH antibody bound to the intracellular PTH protein antigen was examined by ECL (enhanced chemiluminescence) method. That is, the expression level of PTH, CaSR, CHGA, and Cyr61 present in the extracted protein was examined by detecting the degree of ECL reaction by HRP enzyme in the secondary antibody bound to the PTH antibody as the primary antibody.
  • the cells were washed with PBS, and the cells were fixed with 10% formalin solution.
  • the fixed cells were inactivated nonspecific proteins with 2% BSA (bovine serum albumin) solution, and then treated with 2% BSA solution containing primary antibody (PTH, CHGA, Cyr61) to each cell for 4 hours at 37 ° C. Reacted for a while.
  • the unreacted antibody was washed twice with PBS, and then the fluorescent secondary antibody (mouse IgG-FITC, rabbit-IgG-Rhodamine, Invitrogen) corresponding to the primary antibody was included.
  • the 2% BSA solution was reacted at 37 ° C. for 1 hour.
  • DAPI 6-Diamidino-2-Phenylindole, Dihydrochloride
  • Molecular Probes® 6-Diamidino-2-Phenylindole, Dihydrochloride
  • the cell surface was washed twice with PBS, and then the cover glass to which the cells were attached was removed, placed on a glass for fluorescence microscopy, and then attached to the glass with a mounting solution. The expression and intracellular distribution of each antibody on the microscope were examined.
  • the expression pattern of PTH dispersed and distributed in the cytoplasm appears in the form of granules, and the hormonal storage and secretion process is performed through the formation of secretory granules and similar mechanisms like other secretory hormones. It could be predicted that it would come true.
  • the intracellular distribution patterns of CHGA were found not to be expressed in undifferentiated tonsil cells as shown in FIG. 10, but as the differentiation period elapsed in the differentiated tonsil cells, The expression of CHGA was increased and evenly distributed in the cytoplasm.
  • PTH is a major hormone that works to maintain homeostasis of calcium in vivo by recognizing changes in extracellular calcium concentrations. In vivo application and use of PTH produced from parathyroid tissue cells differentiated from tonsil cells, it is important to investigate whether PTH normally performs its own functions.
  • the extracellular calcium concentration was variously controlled to investigate the change of PTH secretion capacity according to the external calcium concentration.
  • differentiation inducing agent was treated with T-MSC for 7 days to induce differentiation into parathyroid tissue.
  • the cells differentiated into parathyroid glands were cultured in culture medium in which the calcium concentration in the cell culture medium was varied at normal concentration (1.5 mM), low concentration (0.09 mM) and high concentration (3.0 mM).
  • the cultured T-MSC was collected for each time period, and the amount of PTH secreted into the cell culture fluid according to the external calcium concentration was measured. It was investigated by ELCIA method.
  • the secretion of PTH was significantly lower than the normal calcium concentration.
  • the differentiated parathyroid tissue can also control the level of PTH secretion according to extracellular calcium concentration, and can control PTH secretion to perform the unique function of PTH, which contributes to maintaining homeostasis of calcium concentration by controlling calcium absorption / resorption. The result shows that there is.
  • the cell appearance of tonsils also shows that a large number of particles of secretory granules are formed, thereby showing that a change in cell morphology according to secretory granulation can also be induced (FIG. 13).
  • CaSR calcium sensitive receptor
  • CaSR is a receptor that exists on the surface of parathyroid cells, and it recognizes the extracellular calcium concentration and plays a role in maintaining and controlling the homeostasis of the calcium concentration in the blood.
  • the calcium concentration is low, it recognizes and promotes the secretion of the stored PTH to the outside of the cell, where the secreted PTH moves to bone tissue through blood circulation and is present on the surface of osteoblasts (PTH receptor, PTHR)
  • PTH receptor PTHR
  • CaSR having such a function is regulated by the amount of the receptor itself. Therefore, the expression level of CaSR and the expression level of PTH were examined using tonsil cells exposed to various extracellular calcium concentrations, and the correlation between the generated PTH and CaSR was investigated.
  • the expression level of CaSR is increased in the low calcium state, and the expression level is significantly decreased in the high calcium state, and CaSR may act as a major factor in the normal homeostasis control process of PTH produced in tonsil cells. I could see that.
  • Example 13 Confirmation of intracellular microstructure of tonsil stem cells differentiated into parathyroid tissue
  • differentiated tissues When stem cells are used to induce differentiation into specific organs, differentiated tissues have structural similarities with actual organ tissues or cells in order to perform specific organ specific functions.
  • amygdala stem cells differentiated into parathyroid tissue was examined by electron microscopy to investigate similarity with normal parathyroid tissue.
  • Electron microscopy was used to investigate changes in intracellular microstructure of T-MSCs differentiated into parathyroid glands.
  • the intracellular microstructure changes were investigated using cells at day 7 of differentiation, the point of maximum PTH secretion.
  • a control group cells on day 7 which were not treated with differentiation-inducing activin A and Shh were used.
  • the cells collected in pellet form were stored in 2.5% glutaraldehyde solution and fixed at 4 ° C.
  • the undifferentiated tonsils showed the basic structure of the nucleus, mitochondria and Golgi like other normal cells (Fig. 15a). ).
  • Osteoblast function by cell culture fluid obtained from T-MSCs differentiated into parathyroid tissue releasing PTH using preosteoblast MC3T3-E1 cells was investigated.
  • the culture medium was replaced every 3-4 days, and treated with various concentrations of cell culture medium (conditioned medium, CM) containing PTH secreted out of the cell (10, 50, 100, 1000 pg in PTH content in the cell culture solution).
  • CM cell culture medium
  • the culture solution is removed, and the cells attached to the bottom of each well are washed with PBS, and then fixed with 60% isopropyl alcohol for 5 minutes, washed twice with distilled water, 2 It was stained for 3 minutes with% Alizarin red S aqueous solution (pH 4.2). After staining was completed, the Alizarin red S aqueous solution was removed and immediately washed three times with distilled water, and then compared with a control group (MC3T3-E1 cultured without inducing differentiation) by observing with a phase contrast microscope.
  • a control group M3T3-E1 cultured without inducing differentiation
  • osteocalcin protein which is a representative bone formation marker
  • the expression of osteocalcin also increased as the concentration of CM increased, which was lower than that of the hormone estrogen. It showed a similar degree of bone formation ability as treated (Fig. 16).
  • PTH produced by the method of the present invention is capable of performing physiological functions unique to PTH when applied in vivo, and thus can be used as a therapeutic agent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Rheumatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne un procédé de préparation d'une cellule souche mésenchymateuse chimérique comprenant une étape consistant à mélanger et à cultiver des cellules souches mésenchymateuses dérivées du tissu amygdalien prélevées sur des donneurs différents, et une population de cellules souches mésenchymateuses chimériques préparée par ledit procédé. De plus, cette invention concerne un procédé pour induire la différenciation de cellules du tissu parathryroïdien des cellules souches dérivées d'amygdales, un procédé de production de parathormones à partir des cellules du tissu parathyroïdien obtenues par le procédé selon l'invention, un agent thérapeutique cellulaire comprenant des cellules de tissu parathyroïdien pour traiter l'hypoparathyroïdisme ou l'ostéoporose ; et une méthode destinée à traiter l'hypoparathyroïdisme ou l'ostéoporose à l'aide dudit agent thérapeutique cellulaire.
PCT/KR2013/003644 2012-04-27 2013-04-26 Population de cellules souches mésenchymateuses chimériques et son procédé de préparation, et procédé de production de parathormones à l'aide de cellules souches dérivées d'amygdales WO2013162330A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2012-0044970 2012-04-27
KR20120044970A KR101508413B1 (ko) 2012-04-27 2012-04-27 키메라 중간엽 줄기세포군 및 그의 제조방법
KR1020130045033A KR101520531B1 (ko) 2013-04-23 2013-04-23 편도줄기세포를 이용하여 부갑상선 호르몬을 생산하는 방법
KR10-2013-0045033 2013-04-23

Publications (1)

Publication Number Publication Date
WO2013162330A1 true WO2013162330A1 (fr) 2013-10-31

Family

ID=49483535

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2013/003644 WO2013162330A1 (fr) 2012-04-27 2013-04-26 Population de cellules souches mésenchymateuses chimériques et son procédé de préparation, et procédé de production de parathormones à l'aide de cellules souches dérivées d'amygdales

Country Status (1)

Country Link
WO (1) WO2013162330A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2581023C1 (ru) * 2015-02-11 2016-04-10 Федеральное государственное бюджетное научное учреждение "Иркутский научный центр хирургии и травматологии" Способ коррекции послеоперационного гипопаратиреоза
CN108990964A (zh) * 2018-08-09 2018-12-14 华东理工大学 细胞冻存液
CN110387350A (zh) * 2018-04-18 2019-10-29 江苏齐氏生物科技有限公司 一种小鼠骨髓间充质干细胞的分离及培养
RU2806978C1 (ru) * 2022-12-27 2023-11-08 Федеральное государственное бюджетное образовательное учреждение высшего образования "Сибирский государственный медицинский университет" Министерства здравоохранения Российской Федерации Способ интратиреоидной аутотрансплантации ткани околощитовидных желез

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060182724A1 (en) * 2005-02-15 2006-08-17 Riordan Neil H Method for expansion of stem cells
KR20090069013A (ko) * 2007-12-24 2009-06-29 동국대학교 산학협력단 골수기원 중간엽 줄기세포를 연골세포로 분화시키는 방법
US20120088272A1 (en) * 2009-06-10 2012-04-12 Bts Research International Pty Ltd Methods of generating hybrid/chimeric cells, and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060182724A1 (en) * 2005-02-15 2006-08-17 Riordan Neil H Method for expansion of stem cells
KR20090069013A (ko) * 2007-12-24 2009-06-29 동국대학교 산학협력단 골수기원 중간엽 줄기세포를 연골세포로 분화시키는 방법
US20120088272A1 (en) * 2009-06-10 2012-04-12 Bts Research International Pty Ltd Methods of generating hybrid/chimeric cells, and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JANJANIN, SASA ET AL.: "Human palatine tonsil: a new potential tissue source of multipotent mesenchymal progenitor cells", ARTHRITIS RESEARCH & THERAPY, vol. 10, no. 4, 28 July 2008 (2008-07-28), pages 1 - 12 *
RYU, KYUNG-HA ET AL.: "Tonsil-derived mesenchymal stromal cells: evaluation of biologic, immunologic and genetic factors for successful banking", CYTOTHERAPY, vol. 14, no. 10, 17 August 2012 (2012-08-17), pages 1193 - 1202 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2581023C1 (ru) * 2015-02-11 2016-04-10 Федеральное государственное бюджетное научное учреждение "Иркутский научный центр хирургии и травматологии" Способ коррекции послеоперационного гипопаратиреоза
CN110387350A (zh) * 2018-04-18 2019-10-29 江苏齐氏生物科技有限公司 一种小鼠骨髓间充质干细胞的分离及培养
CN108990964A (zh) * 2018-08-09 2018-12-14 华东理工大学 细胞冻存液
CN108990964B (zh) * 2018-08-09 2022-01-28 华东理工大学 细胞冻存液
RU2806978C1 (ru) * 2022-12-27 2023-11-08 Федеральное государственное бюджетное образовательное учреждение высшего образования "Сибирский государственный медицинский университет" Министерства здравоохранения Российской Федерации Способ интратиреоидной аутотрансплантации ткани околощитовидных желез
RU2817205C1 (ru) * 2023-04-13 2024-04-11 Государственное бюджетное учреждение здравоохранения города Москвы городская клиническая больница имени С.П. Боткина департамента здравоохранения города Москвы (ГБУЗ ГКБ им. С.П. Боткина ДЗМ) Способ лечения послеоперационного гипопаратиреоза после оперативного вмешательства у больных с вторичным гиперпаратиреозом

Similar Documents

Publication Publication Date Title
WO2013094988A1 (fr) Procédé pour produire des cellules tueuses naturelles, cellules tueuses naturelles produites de cette façon, et composition pour traiter des cancers et des maladies infectieuses contenant les mêmes
WO2012026712A4 (fr) Composition pharmaceutique utilisée dans la prophylaxie ou le traitement de maladies immunitaires ou inflammatoires, contenant des cellules souches traitées par agoniste du nod2 ou un de leurs produits mis en culture
WO2019107913A1 (fr) Procédé d'amélioration de la qualité d'une cellule thérapeutique par la mesure du glutathion en temps réel
WO2011049414A2 (fr) Procédé pour l'induction d'une migration de cellules souches adultes dérivées de tissu adipeux
WO2018124642A9 (fr) Composition pour la différenciation de cellules souches de pulpe dentaire en cellules progénitrices des odontoblastes et anticorps monoclonal de type igg ou igm se liant spécifiquement à la surface de cellules progénitrices des odontoblastes
WO2014181954A1 (fr) Composition de milieu de culture pour améliorer la capacité régénérative de cellules souches, et procédé de culture de cellules souches l'utilisant
WO2019083281A2 (fr) Nouvelle cellule souche musculo-squelettique
WO2021045374A1 (fr) Composition de milieu pour la différenciation d'organoïde hépatique proliférant et procédé de préparation d'organoïde hépatique l'utilisant
WO2013162330A1 (fr) Population de cellules souches mésenchymateuses chimériques et son procédé de préparation, et procédé de production de parathormones à l'aide de cellules souches dérivées d'amygdales
WO2017155166A1 (fr) Procédé de reprogrammation cellulaire utilisant l'imposition d'une transition environnementale médiée par une stimulation physique
WO2022025559A1 (fr) Composition comprenant des exosomes dérivés de cellules souches et sa méthode de production
WO2019216623A1 (fr) Vaccin cellulaire possédant une tolérance immunitaire pour le traitement du diabète et de l'obésité et procédé de production de cellules sécrétrices d'insuline
WO2021033990A1 (fr) Composition comprenant des exosomes dérivés d'un précurseur de cellule souche mésenchymateuse dérivée d'une cellule souche pluripotente induite pour la prévention ou le traitement de la stéatohépatite non alcoolique
WO2022004938A1 (fr) Procédé de préparation de cellules souches de type mésenchymateuses
WO2011102680A2 (fr) Antigène cd49f favorisant la prolifération, la pluripotence et la reprogrammation de cellules souches adultes par l'intermédiaire de la voie pi3k/akt/gsk3
WO2019050350A2 (fr) Cellule de type de sertoli dérivée de cellule souche procédé de préparation associé et utilisation associée
WO2016117960A1 (fr) Cellules souches mésenchymateuses surexprimées par grim19 efficaces dans le traitement d'une maladie immunitaire, et leur utilisation
Wang et al. Immunogenicity and immune modulation of osteogenic differentiated mesenchymal stem cells from Banna minipig inbred line
WO2022030973A1 (fr) Composition thérapeutique cellulaire comprenant des cellules souches à mémoire t dédifférenciées des lymphocytes infiltrant les tumeurs, et méthode de production de la composition
WO2015023165A1 (fr) Composite de régulation d'une inflammation et cellules souches mésenchymateuse stabilisées ayant une fonction optimisée de régulation de l'immunité par blocage de la molécule de signalisation stat3
WO2019059713A2 (fr) Procédé pour la production de cellules tueuses naturelles et son utilisation
WO2018008941A1 (fr) Complexe comprenant un lysat exempt de cellules de cartilage et des cellules souches pour la stimulation de la différenciation du cartilage et son utilisation
WO2021187911A1 (fr) Exosome dérivé de plasma de sang ombilical ou mimétique correspondant et utilisation pharmaceutique correspondante
WO2021091185A1 (fr) Composition pour la culture cellulaire comprenant un facteur paracrine
WO2020122498A1 (fr) Composition pharmaceutique pour le traitement de la pancréatite, comprenant des cellules souches clonales

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13781434

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13781434

Country of ref document: EP

Kind code of ref document: A1