WO2013162330A1 - Chimeric mesenchymal stem cell population and preparation method therefor, and method for producing parathyroid hormone using tonsil-derived stem cells - Google Patents

Abstract

The present invention relates to a method for preparing a chimeric mesenchymal stem cell population comprising a step of mixing and culturing tonsil tissue-derived mesenchymal stem cells obtained from different donors, and a chimeric mesenchymal stem cell population prepared by the method. Additionally, the present invention relates to a method for inducing differentiation of parathyroid tissue cells from tonsil-derived stem cells, a method for producing parathyroid hormones from the parathyroid tissue cells obtained from the method, a cell therapeutic agent including the parathyroid tissue cells for treating hypoparathyroidism or osteoporosis, and a method for treating hypoparathyroidism or osteoporosis using the cell therapeutic agent.

Description

Chimera mesenchymal stem cell population, preparation method thereof and method of producing parathyroid hormone using tonsilocytes

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.

In recent years, studies have been actively conducted on adult stem cells having the ability of organ regeneration and immune response regulation. Sources of adult stem cells used in clinical and research are bone marrow, adipocytes, and cord blood (Ding DC). , et al., Cell Transplant., 20: 5-14, 2011; Lee OK, et al., Blood, 103: 1669-1675, 2004). However, conventional adult stem cells have some limitations in their use, and the process of collecting tissue is invasive, difficult to obtain a sufficient amount, and most of them are tissues of mesodermal origin, and thus are not easy to differentiate into tissues of ectoderm and endoderm origin. (Jung YJ, et al., Stem Cells Dev., 15: 687-695, 2006). To overcome these limitations, research is needed to diversify the source of stem cells (Uranio MF, et al., Mol. Reprod. Dev., 78361L: 373, 2011; Cho KA, et al., Cell Biol. Int , 33: 772-777, 2009; Ivanovski SS, et al., Oral Dis., 12: 358-363, 2006).

On the other hand, 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.

Recently, the results of extracting mesenchymal stem cells (MSC) from the tissues of tonsils have been reported, and the tonsils are attracting attention as a new source of adult stem cells (Janjanin SF, et al., Arthritis Res) Ther., 10: R 83, 2008). Stem cells derived from the tonsils exhibit the same level of stem cell capacity as other adult stem cells, and differentiate into mesodermal cells (adipogenesis, osteocytosis, chondrogenesis). Can be. The advantage of such tonsils is that they are discarded through tonsillectomy, so the supply and demand of materials is relatively smooth.

Parathyroid hormone (PTH) 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.

Under these backgrounds, 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. In addition to showing the same characteristics as the stem cells, even when performing the freezing and thawing process for the mixed cultured stem cells have the same cellular capacity as the stem cells before cryopreservation, confirmed that the proliferation rate of stem cells increases Thus, it was confirmed that 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.

It is still another object of the present invention to provide a cell therapeutic agent for the treatment of parathyroid hypoplasia or osteoporosis comprising 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.

In addition, 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. In addition, by adjusting the extracellular calcium concentration of the parathyroid tissue cells of the present invention, a large amount of parathyroid hormone is secreted out of the cells so that parathyroid hormone can be easily obtained.

1 shows that T-MSCs proliferate in culture under laboratory conditions.

2 shows that T-MSCs express specific surface antigens.

3 shows that T-MSCs can differentiate into mesodermal and endoderm cells. In this case, FoxA2 means forkhead box A2; Sox17 means sex determining region Y-box 17; CXCR4 refers to chemokine (C-X-C motif) receptor 4; Eya1 means eyes absent homolog 1; Six1 means SIX homeobox; Pax1 means paired box 1; BMP4 means bone morphogenic protein 4; GCM2 means glial cells missing homolog 2; CaSR means calcium sensing receptor; CCL21 means chemokine (C-C motif) ligand 21; PTH means parathyroid hormone; GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase.

4 shows that T-MSCs have immunomodulatory activity.

Figure 5 is a diagram illustrating the process of separating the amygdala stem cells from tonsils obtained by tonsillectomy.

6 is a view showing the change in cell shape according to differentiation period from tonsil derived cells.

7 is a view showing the results of the PTH concentration irradiation secreted into the cell culture medium during the differentiation period.

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.

12 is a diagram showing the regulation of PTH secretion according to the extracellular calcium concentration change.

13 is a view showing the change in the cell shape of tonsil stem cells according to the extracellular calcium concentration change.

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.

As one embodiment for achieving the above object, 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.

Specifically, 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.

As used herein, 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. For the purpose of the present invention, the amygdala is used as a source tissue for providing tonsil-derived mesenchymal stem cells.

As used herein, the term "mesenchymal stem cells (MSCs)" 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. For the purposes of the present invention, 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.

As used herein, the term “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. "Totipotent 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.

As used herein, the term "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.

The term "chondrocytes" of the present invention 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.

As used herein, the term "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.

The term "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.

In the method for the mixed culture of tonsil-derived mesenchymal stem cells of the present invention, 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. .

In addition, 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. . At this time, 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.

The term "mixed culture" of the present invention 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.

As another embodiment for achieving the above object, 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.

The term “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. For the purposes of the present invention, 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. 2A), and the immune-related expressing antibodies (HLA-DR, CD40, CD80, CD86) are also negative (FIG. 2B). . In addition, they can be differentiated into mesodermal cells, adipocytes, bone cells and chondrocytes (FIG. 3A), and also into endoderm cells, parathyroid tissue cells (FIG. 3B).

According to one embodiment of the present invention, 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).

As another embodiment for achieving the above object, 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.

The term "frozen" 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. In this case, a solution containing a cryoprotectant may be used to prevent cell damage during freezing.

The term "freeze protection agent" of the present invention 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.

The term "thawing" of the present invention, 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.

After mixing and incubating the mesenchymal stem cells derived from tonsil tissue provided by the present invention, and performing the freezing and thawing process, it was surprisingly found that the proliferative capacity was improved compared to the mesenchymal stem cells that did not perform the freezing and thawing process. 1b).

As another embodiment for achieving the above object, it provides a chimeric mesenchymal stem cell population having improved proliferative capacity prepared by the above method.

The term "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.

According to an embodiment of the present invention, when 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.

As another embodiment for achieving the above object, 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. Cultured in medium to differentiate into chondrocytes, or the chimeric mesenchymal stem cell group was cultured in culture medium containing ascorbic acid 2-phosphate, dexamethasone and β-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.

On the other hand, 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. A method of measuring the change in cell surface labeling or morphology, a method of investigating morphological changes of cells using an optical microscope or a confocal microscope, a method of measuring a change in gene expression profile, and the like. -PCR, Oil red O staining, Safranin O staining, Type II collagen immunohistochemical staining, alkaline phosphate staining and Alizarin S staining can be used.

According to one embodiment of the invention, 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).

As another embodiment for achieving the above object, 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. At this time, 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.

As used herein, the term "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. Or 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. Can be used for 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.

In another aspect of the invention, 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.

Prior to the present invention, studies have been conducted to differentiate parathyroid tissue from some undifferentiated human embryonic stem cells or adult stem cells derived from thymus tissues of children. Never had.

In addition, 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.

However, 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.

In this embodiment, 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.

The term "tonsilocytes" of the present invention 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.

In the method of the present invention, the amygdala stem cells may be collected from tonsil tissue isolated from a patient who has undergone tonsillectomy, but are not limited thereto.

When collected from tonsil tissue isolated from the patient, the collected tonsil is washed with buffer, and then treated with enzyme mixture to decompose the connective tissue. At this time, 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

In the method of the present invention, 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, on the other hand, 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. .

In the method of the present invention, activin A and sonic hedgehog can be purchased by using any commercially available activin A and sonic hedgehog.

In the method of the present invention, when 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.

Preferably, the concentrations of Activin A and Sonic Hedgehog are each 80 ng / ml to 200 ng / ml, most preferably 100 ng / ml each.

According to the method of the present invention, when the amygdala stem cells are cultured in a medium containing activin A and sonic hedgehog at a concentration of 50 to 300 ng / ml, respectively, the parathyroid tissue from the amygdala stem cells may be shortened only by 4 days of culture. Can differentiate cells.

Preferably, according to 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.

In the method of the present invention, 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. Generally, it 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. Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and 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 β-mercaptoethanol.

In a specific embodiment of the present invention, 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. As a result, 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.

In addition, as a result of culturing for three weeks while replacing the culture medium with fresh medium every four days, it was confirmed that the production of parathyroid hormone was significantly increased after 7 days, and GCM2 (glial cells missing homolog 2) specific to parathyroid tissue. , CaSR (calcium sensing receptor), CCL21 (chemokine (CC motif) ligand 21) and the expression of the parathyroid hormone (PTH) genes were confirmed.

In another aspect of the present invention, the present invention provides a method for producing parathyroid hormone using tonsilocytes. Specifically, 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.

As described above with regard to the method of differentiating parathyroid tissue cells from tonsil stem cells, parathyroid tissue cells differentiated from tonsil stem cells according to the method of the present invention 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.

In the method of the present invention, tonsil stem cells, activin A, and sonic hedgehog are as described above.

In the parathyroid hormone production method of the present invention, 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.

On the other hand, 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.

Therefore, in 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.

Preferably, in order to promote secretion of parathyroid hormone from parathyroid tissue 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.

Alternatively, 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. In addition, 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.

In one specific embodiment of the present invention, 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.

In addition, 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. When 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.

As another embodiment of the present invention, the present invention provides parathyroid tissue cells differentiated from tonsil stem cells.

Specifically, 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 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.

Thus, 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.

Accordingly, as another embodiment of the present invention, the present invention provides a cell therapy agent of hypothyroidism or osteoporosis comprising the parathyroid tissue cells of the present invention.

In the present invention, the term "cell therapeutic agent" is as described above.

Diseases in which 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. It is difficult to maintain proper blood calcium level continuously by indirect method, and it is difficult to secure adequacy of treatment effect such as emergency frequently due to hypocalcemia, and to develop gastrointestinal complications such as gastritis due to long-term use of calcium preparations. There are several problems, including this increase. Therefore, instead of taking calcium, when using a cell therapy comprising parathyroid tissue cells capable of producing parathyroid hormone, parathyroid hypoplasia can be treated without side effects.

On the other hand, 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, and as a treatment for osteoporosis, 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. However, 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. .

The term "treatment" in the present invention means any action that improves or benefits the condition of the disease by administration of the cell therapy.

In addition, 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.

In addition, 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.

In the pharmaceutical preparations, in addition to the active ingredient, 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. Preferably, but not limited to engraftment or implantation directly into the disease site of the patient in need of treatment or implantation or injection directly into the abdominal cavity. In addition, 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. In addition to 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 . However, it should be understood that 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.

In another embodiment of the present invention, 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.

As used herein, 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Obtaining Amygdala Mesenchymal Stem Cells

Nine patients under 10 years of age (5 boys and 4 girls, mean age = 6.2 years) underwent otolaryngology-head and neck surgery. It was conducted at Ewha Womans University Mokdong Hospital and passed the deliberation of the Clinical Ethics Committee (ECT 11-53-02). Two-thirds of the extracted tonsils were used for histology and one third were used for the experiment.

5 is a method for isolating and culturing stem cells from the obtained tonsil tissue.

Specifically, the collected tonsils were washed with physiological saline, and the enzyme mixture 210 U / mL collagenase type I (Invitrogen) and 10 μg / 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 ℃ 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. 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 μg / 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.

The 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.

Example 2 Adhesion and Colony-forming Unit-Fibroblast (CFU-F) Analysis

For adhesion and CFU-F analysis of frozen and thawed T-MSCs and frozen T-MSCs, freshly-cultured fresh T-MSCs (FCs) were incubated for 14 days and then cultured. The cells were washed with PBS, fixed in 100% methanol for 5 minutes, then the methanol was removed and dried in air for 5 minutes. Giemsa staining was performed on the dried cells and colonies of fibroblast-like cells were identified using phase contrast microscopy (Castro-Malaspina H, et al., Blood, 56: 289-301, 1980; Baksh D, et al., Exp. Hematol., 31: 723-732, 2003).

In addition, cryopreserved-and-thawed cells (CTCs), which have undergone freezing and thawing, were cultured for 7 days and applied to the same method as described above.

On the other hand, in order to confirm the cell differentiation capacity of FC and CTC, 3 X 10 ³ cells were inoculated into 96-well plates containing culture medium, and cultured with MTT (at 0, 3, 7 and 14 days). MTT assay was performed using 3- (4,5-dimethylthizol-2-yl) 2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich).

As a result, the number of mononuclear cells obtained from each tonsil (2 cm x 1.5 cm x 1.5 cm) was about 10 ⁸ to 10 ⁹ , and FC and CTC each exhibited similar levels of adhesion (FIG. 1). Levels of CFU-F results are shown (FIG. 1A).

In addition, 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). However, mixed CTC cultures obtained from several donors showed relatively high levels of proliferative capacity.

Example 3: Cell Phenotyping Analysis

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.

Specifically, FC and CTC were incubated for 14 days and 7 days, respectively, and stained at 4 ° C. for 30 minutes using respective antibodies (2 μg / ml) capable of reacting with surface antigen and analyzed using the FACSCaliur system. . In this case, 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).

In addition, an IgG1 or IgG2a (Becton Dickinson) isotype antibody, which is an antibody capable of performing nonspecific binding, was used as a control.

As a result, 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).

As a result of comparison with follicular dendritic cells (FDC), which are derived from tonsils and are known to proliferate with adhesion similar to T-MSC, as shown in FIG. 2C, T-MSC is a different cell from FDC. Could.

In addition, there was no difference in phenotype between FC and CTC groups.

Example 4 Confirmation of Differentiation Capacity of T-MSCs into Mesodermal Cells

To determine whether T-MSCs can differentiate into mesodermal cells, T-MSCs were used to induce differentiation into mesodermal cells, adipocytes, bone cells and chondrocytes.

Example 4-1 Differentiation into Adipocytes

Dispense 10 ⁴ T-MSCs per well into a 12-well plate containing adipocyte culture (Invtorgen) containing 3-isobutyl-1-methyl-gentine, dexamethasone, indomethacin and insulin, and add the culture 3- Incubate for 3 weeks with replacement every 4 days. After the incubation was completed, the culture solution was removed, the cells attached to the bottom of each well were washed with PBS, fixed for 5 minutes using 4% paraformaldehyde, each well was dried, and oil red O ( Sigma-Aldrich) was stained at room temperature for 10 minutes. After staining, oil red O was removed and immediately washed 4 times with distilled water, and then compared with a control group (T-MSC incubated without inducing differentiation) by observing with a phase contrast microscope (Yang Z, et al., Methods Mol. Biol., 698: 353-366, 2011; Maxson S, et al., J. Tissue Eng. Regen. Med., 2: 147-154, 2008).

Example 4-2 Differentiation into Osteoblasts

Dispense 10 ⁴ T-MSCs per well into 12-well plates containing bone cell culture (Invtorgen) containing ascorbic acid 2-phosphate, dexamethasone and β-glycerophosphate, and replace the culture every 3-4 days. Incubated for 3 weeks. After the incubation was terminated, the culture solution was removed, the cells attached to the bottom of each well were washed with PBS, fixed for 5 minutes using 60% isopropyl alcohol, washed twice with distilled water, 2% Alizarin Staining was performed with a red O aqueous solution (pH 4.2) for 3 minutes. After staining was completed, the Alizarin red O aqueous solution was removed and immediately washed three times with distilled water, and then compared with a control group (T-MSC cultured without inducing differentiation) by observation under a phase contrast microscope (Yang Z, et al., Methods Mol. Biol., 698: 353-366, 2011; Maxson S, et al., J. Tissue Eng. Regen. Med., 2: 147-154, 2008).

Example 4-3 Differentiation into Chondrocytes

2 x 10 ⁶ 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 μm 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. Sulfated preteoglycans contained in the cell sections fixed on the slide glass were stained with Alcian blue (pH 2.3), and compared with a control group (T-MSC cultured without inducing differentiation) under a microscope. (Yang Z, et al., Methods Mol. Biol., 698: 353-366, 2011).

Example 4-4: Results

After 3 weeks of induction of fat differentiation, it was confirmed that intracellular lipid droplets were formed (FIG. 3A, left), and differentiation into bone cells and deposition of calcium occurred (FIG. 3A, center). Differentiation of the furnace and accumulation of matrix sulfated preteoglycan was confirmed (FIG. 3A, right). In contrast, no differentiation-specific characteristics were observed in the control group.

Example 5 Confirmation of Differentiation Ability of T-MSCs into Endoderm Cells

To determine whether T-MSCs can differentiate into endoderm cells, T-MSCs were induced to differentiate into parathyroid cell-like cells, which are endoderm cells.

Specifically, 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. GCM2 (glial cells missing homolog 2), CaSR (calcium sensing receptor), and CCL21 specific for parathyroid tissue cells (chemokine (CC motif) ligand 21) and the expression of parathyroid hormone (PTH) genes were confirmed (Bingham EL, et al., Stem Cells Dev., 18: 1071-1080, 2009).

In addition, total RNA was extracted from endoderm cells differentiated from T-MSC, and cDNA was synthesized by applying 1 μg of the extracted RNA to an RT system (Promega, Madison, Wis.). At this time, 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).

Table 1

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

As a result, the expression of FoxA2, Sox17 and CXCR4, which are marker genes of endoderm cells, was confirmed in RT-PCR results (Fig. 3b), and thymus and parathyroid markers (thymus / parathyroid primordium markers) Eya1, Six1, Pax1, Noggin Expression of BMP4 was also confirmed, and expression of GCM2, CaSR, CCL21 and PTH, which are crucial markers of the parathyroid gland, was also confirmed.

The results show that T-MSCs can be differentiated into parathyroid tissue cells, which are endoderm cells.

Example 6: Analysis of immunomodulatory capacity of T-MSCs

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.

In accordance with known methods, inhibition of PMA-stimulated T cell division was measured (Rasmusson I, et al., Exp. Cell Res., 305: 33-41, 2005).

Using CellTraceTM CFSE Cell Proliferation Kit (Invitrogen), CD4 + T cells were labeled with carboxyfluorescein diacetate (succinimidyl ester) (CFSE). 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 μM 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.

As a result, PMA induced the proliferation of CD4 + T cells in a proportional manner to its administration, but it was confirmed that the induction of proliferation of CD4 + T cells by PMA was inhibited by treatment with T-MSC. Similar levels were shown (FIG. 4A).

Example 7: Short tandem repeats (STR) analysis of T-MSCs extracted from multiple donors

Each T-MSC from three independent donors (two boys and one girl) 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.

Through STR analysis, chimeras (heterozygote) were formed at three sites of D8S1179, D3S1358 and D18S51 among the total 15 sites (FIG. 4B). As shown in the results, it was confirmed that 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.

Example 8: Induction of Differentiation of Tonsil Stem Cells into Parathyroid Tissues

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.

As a result of the culture, as the differentiation period elapsed, the amygdala stem cells formed a multi-layer accumulating into several layers as shown in FIG.

Example 9 Investigation of PTH Secretion by Differentiation Period

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 μm 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.

As a result, as shown in Fig. 7a, 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. As a result of the investigation by differentiation period, it was found that 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). As a result of investigating the cumulative PTH concentration value, the cumulative PTH secretion after 10 days after the differentiation induction solution was investigated at a concentration of 100 pg / ml, 100 pg / ml when continuously cultured differentiated tonsil cells It can be seen that the PTH of can also be obtained (Fig. 7c).

Example 10 PTH Protein and Related Protein Expression by Western Blot Method

After the differentiation step, 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.

10-12% SDS (sodium deoxycholate) -polyacrylamide gel (SDS-PAGE) was prepared, and 30 ㎍ 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.

Of the proteins of each size delivered on the membrane, only the membrane portion corresponding to the PTH protein size is taken, and the PTH specific antibody (Ab frontier, LF-MA0140), calcium sensing receptor (CaSR, Abcam ab18200) , Chromagranin A (CHGA, AVIVA ARP-41930-T100), Cystein rich protein 61 (Cyr61, Santa Cruz SC-13100) and osteocalcin (OC, Santa Cruz SC-74495 ) Was reacted with the antibody at 4 ° C. low temperature for 14-16 hours. After the reaction with the primary antibody (diluted at 1: 1000 ratio), 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.

At the same time, the same amount of protein was used for each sample, and the expression level using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody in the protein extracted at each step was verified to demonstrate that the entire western blot method was performed consistently. Investigate.

The expression level of each specific protein was quantitatively analyzed using the Image J program.

As a result, as shown in Figure 8, as the differentiation period, the expression of PTH protein in tonsil cells increased. In particular, after 4 days of eruption induction, it was found that significantly increased until 7 days.

Example 11: Investigation of PTH and PTH Secretory Granular Protein Distribution Using Confocal Microscopy

The intracellular distribution of proteins related to PTH and PTH secretion control in T-MSC cells during the differentiation of parathyroid tissue was investigated using a confocal lazer scanning microscope (LSM-5 Pascal EXCITER, Carl Zeiss). .

In order to process the cells to be used for confocal microscopy, a variety of undifferentiated and differentiated cells were prepared at various differentiation periods (7, 14, 21 days), and cover-glass was prepared at the bottom of each well in advance. 10 ⁴ aliquots were dispensed in each well plate.

After differentiation, 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. After reaction with the primary antibody, 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. At this time, DAPI (4 ', 6-Diamidino-2-Phenylindole, Dihydrochloride) (Molecular Probes®), which is specifically stained in the nucleus, was added to simultaneously stain the nuclei in each cell. After completion of the reaction with the secondary antibody, 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.

As a result, as shown in Figure 9, as a result of the investigation during the total differentiation, the expression of intracellular PTH was significantly changed in PTH distribution in the cell group (labeled as Control cell, C) not treated with differentiation-inducing fluid as shown below It was investigated not to appear. On the other hand, when the differentiation induction solution (differentiated cells, denoted as D), the expression of PTH was significantly increased as the differentiation period elapsed. The pattern showed a significantly higher expression distribution at 14 days, showing the same trend as the expression pattern of intracellular PTH protein and the pattern of changes in secreted PTH concentration.

In addition, 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.

In relation to the distribution of PTH in the cells, 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.

As a result of double staining of PTH protein and CHGA at the same time, it was found that the expression sites of PTH and CHGA matched, indicating that there is a direct correlation with CHGA in the process of storing and secreting the generated PTH (Fig. 11).

Example 12: Investigation of PTH Secretion Capacity According to Extracellular Calcium Concentration

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.

Therefore, after the amygdala was differentiated into parathyroid tissue, the extracellular calcium concentration was variously controlled to investigate the change of PTH secretion capacity according to the external calcium concentration.

First, differentiation inducing agent was treated with T-MSC for 7 days to induce differentiation into parathyroid tissue. After inducing differentiation for 7 days, 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). After exposing the cultured T-MSC to a culture solution containing various calcium concentrations for 3, 8, 24, and 48 hours, the cell culture fluid 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.

As a result, as shown in Figure 12, in the low calcium state showed a high concentration of PTH secretion compared to the normal calcium concentration, in particular, the secretion after 24 hours exposure was further increased. On the other hand, in the high calcium state, 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.

Particularly, in the low calcium state, 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).

In addition, a factor known to recognize extracellular calcium concentrations and to control the secretion of stored PTH is calcium sensitive receptor (CaSR). 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. When 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) In combination with the reaction, the stored calcium is released into the blood to maintain the homeostasis of calcium in the blood occurs in vivo. It is known that 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.

As a result, as shown in Fig. 14, 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

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.

In this regard, the intracellular microstructure of 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. At this time, as a control group, cells on day 7 which were not treated with differentiation-inducing activin A and Shh were used. After washing the cell surface of the control group (Control, C) and the 7th day of differentiation (Differentiation, D) using PBS, remove the attached cells by treatment with trypsin, and then using a centrifuge at 5 Centrifuged for a minute to collect the fallen cells. The cells collected in pellet form were stored in 2.5% glutaraldehyde solution and fixed at 4 ° C.

In order to observe the intracellular microstructure, it was examined using a transmission electron microscope (MODEL H-7650 (2006), H-600 (1982) / HITACHI). The observed magnification was 4,000 times and 12,000 times.

Comparing and observing undifferentiated tonsils and parathyroid tonsils, the undifferentiated tonsils showed the basic structure of the nucleus, mitochondria and Golgi like other normal cells (Fig. 15a). ).

On the other hand, in the cells induced by differentiation of the parathyroid gland, it was investigated that in addition to the basic cellular microstructures, a number of structures regarded as cell granules were formed (FIG. 15B). In addition, a structure similar to that of the pro-secretory granule (pro-secretory granule) was also observed (FIG. 15b), and structurally confirmed that the differentiation into the hormone-secreting tissue secreting PTH proceeded well.

Example 14: Osteoblast function investigation using progenitor osteoblasts

Since PTH has been reported to have bone formation ability through the PTH receptor (PTHR) on the surface of osteoblasts, the role of PTH itself as a new osteoporosis treatment through direct induction of bone formation ability has been suggested.

Based on this, we investigated whether PTH produced and secreted from amygdala stem cells differentiated into parathyroid tissues actually has PTH-specific bone formation ability.

Osteoblast function by cell culture fluid obtained from T-MSCs differentiated into parathyroid tissue releasing PTH using preosteoblast MC3T3-E1 cells. That is, MC3T3-E1 cells were seeded 10 ⁴ per each well in 24-well plates, and processes each per well biksan ascorbyl 2-phosphate, dexamethasone and a bone cell culture medium containing a phosphate β- glycerophosphate (Invtorgen) 2 jugan Osteopathic function 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). / ml), the osteogenic ability of secreted PTH was investigated. We compared and compared 17-beta estradiol (10 μM) in clinical trials with PTH and osteoporosis hormone therapy synthesized and marketed by genetic recombination method.

To investigate the osteogenic ability, Western blot analysis was performed using the Alizarin red S method, which stains the cytoplasmic expression and cytoplasmic calcium accumulation of osteocalcin (OC) protein, a representative marker of bone formation.

That is, after the incubation is completed, 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.

As a result of examining the expression level of 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). In addition, compared to the case of treatment with the commercial PTH (104 ng / ml), even when treated with a CM (1000 pg / ml = 1 ng / ml) at a concentration corresponding to 1/100 of the commercial PTH concentration It showed similar bone formation ability, and it was confirmed that PTH produced and secreted in tonsil stem cells has high physiological activity.

As a result of staining the intracellular calcified portion using Alizarin red-S staining, bone formation was induced by CM, and the calcified portion was markedly increased.

The results show that 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.

All data are expressed as mean ± standard deviation (S.D.), and statistically significant tests were analyzed by Student's t-test using GRAPHPAD PRISM software (GraphPad Software Inc., San Diego, Calif.). In all analyzes, cases with P <0.05 were considered statistically significant. All experiments were performed at least three times.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (34)

1. (i) obtaining mononuclear cells from each tonsil isolated from a separate donor;

   (ii) culturing the obtained mononuclear cells to select mesenchymal stem cells; And,

   (iii) mixing the selected mesenchymal stem cells and culturing, the method for producing a chimeric mesenchymal stem cell group.
2. A chimeric mesenchymal stem cell group prepared by the method of claim 1 and exhibiting the following characteristics:

   (i) comprises mesenchymal stem cells isolated from separate tonsils 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) can differentiate into endoderm cells.
3. The method of claim 2,

   The immunologically expressing antibody is HLA-DR, CD40, CD80, CD86 or a combination thereof chimeric mesenchymal stem cell population.
4. The method of claim 2,

   The mesoderm cells are adipocytes, bone cells, chondrocytes or a combination thereof chimeric mesenchymal stem cell group.
5. The method of claim 2,

   The endoderm cells are parathyroid tissue cells chimeric mesenchymal stem cell group.
6. The method of producing a chimeric mesenchymal stem cell group having improved proliferative capacity, comprising freezing and thawing the chimeric mesenchymal stem cell group of claim 2.
7. The method of claim 6,

   Wherein the freezing is performed using a cooling medium, a cryoprotectant or a combination thereof.
8. The method of claim 7, wherein

   The cooling medium is liquid nitrogen.
9. The method of claim 7, wherein

   The cryoprotectant is glycerol, sugar, glucose or a combination thereof.
10. A chimeric mesenchymal stem cell group prepared by the method of claim 6 and having an improved proliferative capacity having the following characteristics:

    (i) exhibits the same level of differentiation as the chimeric mesenchymal stem cell population that did not undergo freezing and thawing; And,

    (ii) show an enhanced level of proliferative capacity than the chimeric mesenchymal stem cell population that did not perform freezing and thawing.
11. The chimeric mesenchymal stem cell group of claim 2 or the chimeric mesenchymal stem cell group having improved proliferative capacity of claim 10 are cultured in chondrocyte differentiation medium containing dexamethasone, ascorbic acid, L-proline, TGF-3, BMP-6 and ITS. Chimera mesenchymal stem cell group comprising the step of differentiating into chondrocytes.
12. A chimeric mesenchymal stem cell group according to claim 2 or a chimeric mesenchymal stem cell group having improved proliferative capacity according to claim 10, comprising a step of culturing a chimeric mesenchymal stem cell comprising a culture medium containing ascorbic acid 2-phosphate, dexamethasone and β-glycerophosphate. Method of differentiating cell population into osteocytes.
13. The method of claim 2, comprising the step of culturing the chimeric mesenchymal stem cell group of claim 2 or the chimeric mesenchymal stem cell group having improved proliferative capacity of claim 10 in a culture medium containing 3-isobutyl-1-methyl-gentine, dexamethasone, indomethacin and insulin. Method of differentiating chimeric mesenchymal stem cell population to adipocytes.
14. Cultivating the chimeric mesenchymal stem cell group of claim 2 or the chimeric mesenchymal stem cell group having improved proliferative capacity of claim 10 in a culture medium containing fetal bovine serum (FBS), activin A (activin A) and Shh (sonic hedgehog) Method for differentiating chimeric mesenchymal stem cell group comprising a parathyroid tissue cells.
15. A cell therapeutic agent comprising stem cells isolated from the chimeric mesenchymal stem cell group of claim 2 or the chimeric mesenchymal stem cell group having improved proliferative capacity of claim 10, or cells differentiated from the chimeric mesenchymal stem cell group.
16. A method of differentiating parathyroid tissue cells from tonsils, comprising culturing tonsils in a medium comprising Activin A and Sonic hedgehog (Shh).
17. The method of claim 16, wherein the activin A and sonic hedgehog are each present in the medium at a concentration of 50 ng / ml to 300 ng / ml.
18. 18. The method of claim 17, wherein the activin A and sonic hedgehog are each present in the medium at a concentration of 100 ng / ml.
19. The method of claim 16, wherein the medium is Dulbecco's modified Eagle medium (DMEM) medium.
20. The method of claim 16, wherein the culturing is carried out for at least 4 days.
21. The method of claim 16, wherein the amygdala stem cells are mesenchymal stem cells.
22. The method of claim 16, further comprising the step of replacing with fresh medium every 4 to 5 days if the cultivation is for 4 days or more.
23. i) culturing tonsilocytes in a medium comprising Activin A and Sonic hedgehog (Shh) to differentiate into parathyroid tissue cells and ii) culture or cell lysis obtained in step i) A method of producing parathyroid hormone using tonsilocytes, comprising the step of separating parathyroid hormone from fluid.
24. The method of claim 23, further comprising, after step i) adjusting the calcium concentration of the culture to secrete parathyroid hormone out of the cell.
25. The method of claim 24 further comprising, after step i), adjusting the calcium concentration of the culture to 0.5 mM or less.
26. The method of claim 23, wherein the activin A and sonic hedgehog are each present in the medium at a concentration of 50 ng / ml to 300 ng / ml.
27. The method of claim 23, wherein the activin A and sonic hedgehog are each present in the medium at a concentration of 100 ng / ml.
28. The method of claim 23, wherein the culturing is carried out for at least 4 days.
29. The method of claim 23, wherein the culturing is carried out for at least 7 days.
30. Parathyroid tissue cells differentiated from tonsil stem cells.
31. The parathyroid tissue cell according to claim 30, which is differentiated by the method of any one of claims 16 to 22.
32. 32. The parathyroid tissue cell of claim 30 which produces parathyroid hormone.
33. A cell therapeutic agent for the treatment of hypothyroidism or osteoporosis comprising parathyroid tissue cells differentiated from tonsil stem cells.
34. A method of treating parathyroid hypoplasia or osteoporosis, comprising administering a cell therapy comprising parathyroid tissue cells differentiated from tonsil stem cells to an individual suspected of hypothyroidism or osteoporosis.

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