KR20140126626A - Method of producing parathyroid hormone from tonsil-derived mesenchymal stem cell - Google Patents

Abstract

The present invention relates to a method for inducing differentiation of a parathyroid tissue cell from a tonsil-derived stem cell, to a method for producing parathyroid hormone from the parathyroid tissue cell obtained by the method, to a cellular therapeutic agent containing the parathyroid tissue cell for treating hypoparathyroidism or osteoporosis, and to a method for treating hypoparathyroidism or osteoporosis, using the cellular therapeutic agent.

Description

Methods for producing parathyroid hormone from tonsil-derived mesenchymal stem cells.

The present invention relates to a method for inducing the differentiation of parathyroid tissue cells from mononuclear stem cells and a method for producing parathyroid hormone from the parathyroid tissue cells obtained by the method.

In recent years, studies on adult stem cells having regeneration and immune responses have been actively conducted. Current sources of adult stem cells used in clinical studies include bone marrow, adipocytes, 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 the tissues is invasive, and it is difficult to obtain a sufficient amount, and most of them are mesodermal origin tissues and it is not easy to differentiate into ectodermal and endodermal origin tissues . To overcome these limitations, it is necessary to study the diversity of stem cell sources.

On the other hand, the human tonsil is a kind of lymphoid epithelium that is present in the human oropharynx of the oropharynx, and its immune function is continued until adolescence, and gradually becomes degenerated, so that the tonsil is abnormally uneven (adenotonsillar hyperplasia) , Tonsillitis (tonsilitis), etc. If you have problems such as tonsillectomy (tonsillectomy) will be removed through.

Recently, studies on the extraction of mesenchymal stem cells (MSCs) from tissues of tonsils have been reported, and the tonsil tissue has been attracting attention as a new source of adult stem cells (Janjanin SF, et al., Arthritis Res Ther., 10: R83, 2008). The stem cells derived from the tonsil tissue show the same level of stem cell function as other adult stem cells, and they are differentiated into mesodermal cells (adipogenesis, osteogenesis, chondrogenesis) But it is not known about the differentiation into endoderm cells. The advantage of such a one-way tissue is that it is an organization that is discarded through tonsillectomy, so the supply and demand of materials is relatively smooth.

On the other hand, parathyroid hormone (PTH) is a hormone composed of 84 amino acids secreted from the parathyroid gland. It is a major hormone that regulates calcium concentration in vivo and can be used as a therapeutic agent for hypoparathyroidism and osteoporosis. However, there has not yet been developed a method to effectively produce parathyroid hormone.

Under these circumstances, the present inventors have made intensive efforts to search for a tissue capable of differentiating from a mononuclear stem cell, and as a result, it has been confirmed that parathyroid tissue cells can be differentiated from mononuclear stem cells and that parathyroid hormone is normally produced and secreted, .

It is an object of the present invention to provide a method of differentiating parathyroid tissue cells from tonsillar stem cells.

It is another object of the present invention to provide a method for producing parathyroid hormone using a somatic stem cell.

It is yet another object of the present invention to provide a parathyroid tissue cell differentiated from a tonsil stem cell.

It is still another object of the present invention to provide a cell therapy agent for treating hypoparathyroidism or osteoporosis comprising parathyroid tissue cells differentiated from tonsil stem cells.

It is still another object of the present invention to provide a method for treating hypoparathyroidism or osteoporosis, which comprises administering a cell therapy agent containing the parathyroid tissue cells of the present invention to a subject suspected of hypoparathyroidism or osteoporosis.

In one aspect to achieve the above object, the present invention provides a method for culturing a somatic stem cell, comprising culturing a somatic stem cell in a medium containing an activin A and a sonic hedgehog (Shh) A method for differentiating parathyroid tissue cells from a cell is provided.

Prior to the present invention, studies have been made to differentiate parathyroid tissue from adult stem cells derived from some undifferentiated human embryonic stem cells or from thymus tissues of pediatric patients. However, studies on the method of differentiating parathyroid tissue from tonsil stem cells have been made There was no enemy.

In addition, when inducing parathyroid tissue from adult stem cells derived from embryonic stem cells or thymus, it takes a long time to induce the parathyroid tissue for 6 months or longer.

However, in the present invention, for the first time, a method of inducing differentiation of parathyroid tissue from a stromal stem cell, which has not been considered as a potential candidate for differentiating into parathyroid tissue, can be obtained within a short period of time.

The term "one-way" in the present invention means a tissue which is located in the back of the neck and the back of the nose and defends the body primarily from substances such as bacteria invading from outside and acts as a lymphocyte immunity do. The one-way includes the pharyngeal one, the ear canal one-way, the oral reconstruction, the tongue one way, and the like. In the present invention, the one-way arrow is used as a source tissue for providing a mesenchymal-derived mesenchymal stem cell.

The term "somatic stem cells" of the present invention refers to mesenchymal stem cells (MSCs) derived from tonsil, and mesenchymal stem cells are differentiated into bone, cartilage, fat, bone marrow, , Which is usually found in the bone marrow, but it also exists in cord blood, peripheral blood, and other tissues.

The term "stem cell" of the present invention means a cell capable of self-replicating and capable of differentiating into two or more new cells, and includes totipotent stem cells, pluripotent stem cells, , And multipotent stem cells (multifunctional stem cells). Totipotent stem cells are pluripotent stem cells that can develop into one complete organism. These cells have the properties of oocyte and spermatozoa until 8th gestation. These cells are separated from the uterus Which means a cell that can develop into one complete organism. Pluripotent stem cells are cells that can occur in various cells and tissues derived from ectoderm, mesoderm, and endodermal layer. It is a cell that is located inside the blastocyst after 4-5 days of fertilization (inner cell mass, which refers to a cell that is known as an embryonic stem cell and that is differentiated into various tissue cells but does not form new life forms.

The term "parathyroid tissue cells" of the present invention means cells constituting the parathyroid gland, an endocrine organs attached to the thyroid gland, secretes parathyroid hormone (PTH), and regulates calcium and phosphorus metabolism in body fluids. In the present invention, it may be contained in parathyroid tissue cells as long as it secretes parathyroid hormone.

The method of differentiating parathyroid tissue cells according to the present invention comprises culturing a somatic stem cell in a medium containing actin A and sonic hedgehog (Shh) at a concentration of 50 ng / ml to 300 ng / ml, respectively, The incubation period of the cells is preferably 4 days or more, more preferably 7 days or more.

In the method of the present invention, the tonsillar stem cells may be collected from the tonsil tissue isolated from patients suffering from the tonsillectomy, but the present invention is not limited thereto.

When taken from a patient's tonsil tissue, the collected tonsil tissue is washed with a buffer, and the enzyme mixture is treated to decompose the connective tissue. Then, the suspended tissue is centrifuged and the cell mixture in the cut tissue is treated with Ficol Only the precipitated portion of the lower layer was cultured using the cell fractionation method and the centrifugation method, and only the cell portion exhibiting adherence to the cell bottom was isolated, and then immunochemistry using cell surface markers among cells showing adherence to the bottom of the culture dish Can be used to separate mononuclear stem cells.

Actin A, which is used as a differentiation inducer in the method of the present invention, is a member of the TGF-beta superfamily and was first known as a protein that promotes FSH release. However, actinib A has been known to induce mesodermal induction, neuronal differentiation, bone remodeling, It is known to have broad biological activity.

Sonic hedgehog protein, on the other hand, is one of three proteins in the mammalian signaling pathway family called hedgehog, and plays a key role in vertebrate organogenesis regulation, such as finger growth and brain organization in the extremities. Sonic hedgehog is known to be a type of morphogen that has a different effect on the cells of the embryo that is developing according to its concentration, and also controls the cell division of adult stem cells and is involved in the development of some cancers .

In the method of the present invention, Actibin A and Sonic hedgehog can be purchased by purchasing any commercially available Actibin A and Sonic hedgehog.

In the method of the present invention, when the concentration of actin A and sonic hedgehog is less than 50 ng / ml, the period required for the differentiation of parathyroid tissue from stromal stem cells is prolonged to several months or the efficiency of differentiation is low. When the concentration of the differentiation inducing agent exceeds 300 ng / ml, there is no improvement in the efficiency of differentiation induction, and it is inefficient to use such a high concentration of the chemical substance.

Preferably, the concentrations of actin A and sonic hedgehog are 80 ng / ml to 200 ng / ml, respectively, most preferably 100 ng / ml each.

According to the method of the present invention, when cultured mononuclear stem cells are cultured in medium containing actin A and sonic hedgehog at concentrations of 50 to 300 ng / ml, respectively, culturing of parathyroid tissue Cells can be differentiated.

Preferably, the method according to the present invention induces differentiation of the parathyroid tissue cells by culturing the somatic stem cells for 4 to 25 days in a medium containing the actin A and the sonic hedgehog at a concentration of 100 ng / ml, respectively If the incubation period exceeds 4 days, the medium can be replaced with fresh medium containing actin A and sonic hedgehog every 4-5 days.

In the method of the present invention, the medium used for inducing differentiation may be a conventional medium that can be used for culturing stem cells, for example, DMEM (Dulbecco's modified Eagle medium), Keratinocyte-SFM free medium, RPMI-1640 and the like, preferably DMEM medium.

The stem cell medium may be supplemented with an additive. (E. G., Serum, e. G., FBS, serum replacement, albumin, or essential amino acids and non-essential amino acids such as glutamine) in an isotonic solution. Furthermore, it has been found that lipid (fatty acid, cholesterol, serum HDL or LDL extract) and other components found in most types of storage medium of this kind (such as insulin or transferrin, nucleoside or nucleotide, pyruvate, G., Glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as? -Mercaptoethanol).

In one specific embodiment of the present invention, when the somatic stem cells were cultured in a medium containing 100 ng / ml of each of Actin A and Sonic hedgehog, 4 days after the culture, the parathyroid hormone The results of the electron microscopic study showed that the differentiated cells had cell granule structure and granule precursor structure in addition to basic cell microstructure, .

In addition, the culture medium was replaced with a fresh medium every 4 days, and the culture was continued for 3 weeks. As a result, the production of parathyroid hormone was remarkably increased from day 7, and GCM2 (glial cells missing homolog 2) , CaSR (calcium sensing receptor), CCL21 (chemokine (CC motif) ligand 21), and PTH (parathyroid hormone)

In another aspect of the present invention, the present invention provides a method for producing parathyroid hormone using a somatic stem cell. Specifically, the present invention relates to a method for culturing a stromal stem cell in a medium containing i) an activin A and a sonic hedgehog (Shh) to differentiate into parathyroid tissue cells, and ii) And separating the parathyroid hormone from the culture solution or the cell lysate obtained in the step (a) of the present invention.

As described above with respect to the method of differentiating the parathyroid tissue cells from the one-way stem cells, the parathyroid tissue cells differentiated from the one-way stem cells according to the method of the present invention produce and secrete parathyroid hormone like normal parathyroid tissue cells. Thus, the parathyroid hormone can be obtained by separating the parathyroid hormone from the culture solution or cell lysate of the parathyroid tissue cells differentiated from the somatic stem cells.

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

In the method for producing parathyroid hormone of the present invention, the step of culturing the somatic stem cells in a culture medium containing actin A and sonic hedgehog and differentiating them into parathyroid tissue cells is the same as described above.

The step of separating the parathyroid hormone from the culture medium or the cell lysate may be carried out using a general method which can be used for the separation and purification of proteins, for example, affinity chromatography, ion exchange chromatography, co-precipitation But the present invention is not limited thereto and can be appropriately selected and carried out by a person skilled in the art.

On the other hand, parathyroid hormone is produced in the parathyroid tissue cells, and then it is present in the form of secretory granule in the cell, and the secretion of extracellular calcium is determined according to the extracellular calcium concentration.

Therefore, in the method of the present invention, in order to facilitate the secretion of the parathyroid hormone produced by the parathyroid tissue cells derived from the somatic stem cells and to facilitate the isolation of the parathyroid hormone, Thereafter, the calcium concentration of the culture medium may be adjusted to secrete the parathyroid hormone out of the cells.

Preferably, the calcium concentration of the culture medium may be adjusted to less than 1.5 mM to promote secretion of parathyroid hormone from the parathyroid tissue cells.

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 isolated from the lysate by dissolving the cells without releasing the parathyroid hormone outside the cell.

The method of producing parathyroid hormone of the present invention is a method for producing parathyroid hormone which can be usefully used for the treatment of hypoparathyroidism or osteoporosis using mononuclear stem cells which can be obtained from a tonsil tissue discarded after a tonsillectomy In addition, by regulating the calcium concentration outside the cells, the secretion of the parathyroid hormone is promoted so that a large amount of hormone can be easily obtained.

In one specific embodiment of the present invention, the expression of the parathyroid hormone was observed during the induction of the differentiation of the parathyroid tissue by culturing the somatic stem cells in a medium containing actin A and sonic hedgehog. As a result, It was confirmed that the expression of parathyroid hormone was significantly increased from day 1 after the induction of differentiation, and that the expression level significantly increased from 7 days after the induction of differentiation. Thus, by the method of the present invention, It was confirmed that the cells can produce parathyroid hormone.

In addition, it was confirmed that the cumulative secretion amount of parathyroid hormone also increases with the lapse of differentiation induction period, and it was confirmed that a large amount of parathyroid hormone can be obtained when the parathyroid tissue differentiated from the somatic stem cells is continuously cultured. It is also confirmed that when the external calcium concentration is lower than the normal concentration (1.5 mM), the secretion of the parathyroid gland hormone is excreted outside the cell, and that the secretion of the parathyroid gland hormone can be controlled by controlling the calcium concentration of the culture fluid when inducing the differentiation of the parathyroid tissue cell Respectively.

In another embodiment of the present invention, the present invention provides a parathyroid tissue cell differentiated from a tonsil stem cell.

Specifically, the parathyroid tissue cells of the present invention can be obtained by the method of differentiating parathyroid tissue cells, which comprises culturing the somatic stem cells as described above in a medium containing actin A and sonic hedgehog, Can be obtained by culturing the mononuclear stem cells in a medium containing the concentrations of actin A and sonic hedgehog at a concentration of 50 ng / ml to 300 ng / ml, respectively, for at least 4 days, and the most preferable method for obtaining the parathyroid tissue cells The concentrations of actin A and sonic hedgehog were 100 ng / ml, respectively.

According to the differentiation method of the present invention as described above, the parathyroid tissue cells obtained by differentiating from the somatic stem cells can produce parathyroid hormone and can regulate the secretion of the parathyroid hormone according to the change of the calcium concentration outside the cells, When the calcium concentration is lower than the normal concentration (1.5 mM), the parathyroid hormone is secreted out of the cell, and when the calcium concentration outside the cell is higher than the normal concentration, the secretion of the parathyroid hormone can be suppressed.

Therefore, the parathyroid tissue cells of the present invention can function as parathyroid tissue cells as well as parathyroid tissue cells present in the body, and can be treated by diseases caused by parathyroid tissue function or parathyroid hormone deficiency or parathyroid hormone And can be usefully used for treatment of known osteoporosis and the like.

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

In the present invention, the term "cell therapeutic agent" is a medicament (US FDA regulation) used for treatment, diagnosis and prevention of cells and tissues prepared by separation, culture and special manipulation from an individual, Diagnosis, and prevention through a series of actions, such as alive, homologous, or xenogeneic cell proliferation screening or other ways to alter the biological characteristics of a cell to restore the function of the cell.

Examples of diseases for which the cell therapy agent containing the parathyroid tissue cells of the present invention can be used include hypoparathyroidism, or osteoporosis.

Parathyroid gland hypothyroidism is a disease in which hypocalcemia occurs due to hypocalcemia caused by low blood calcium due to parathyroid hormone dysfunction. The only way to treat it is by taking excessive calcium, but taking calcium in excess of parathyroid hormone Indirect methods are difficult to maintain adequate blood calcium concentration and it is difficult to maintain the appropriate therapeutic effect because of the frequent emergency situation due to hypocalcemia. And the like. Therefore, when a cell therapy agent containing a parathyroid tissue cell capable of producing parathyroid hormone can be used instead of the calcium administration, it is possible to treat hypoparathyroidism without side effects.

On the other hand, osteoporosis refers to a state in which the amount of bone is reduced and the strength of the bone is weakened due to a qualitative change, and fracture is likely to occur. As a therapeutic agent for osteoporosis, The use of parathyroid hormone has been suggested as a direct promoter of osteogenesis. However, when using the parathyroid hormone, it is impossible to use only the short term (maximum 2 years, US FDA), expensive, daily injections like insulin injection, and rapid bone loss after the treatment It has been reported. Therefore, it is possible to treat osteoporosis without side effects by using a cell therapy agent containing parathyroid tissue cells, which can produce parathyroid hormone rather than administering the parathyroid hormone and can regulate hormone secretion according to the change of the extracellular calcium concentration .

In the present invention, the term "treatment" means any action that improves or alleviates symptoms of the disease upon administration of the cell therapy agent.

In addition, the cell treatment 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 1 ml can do.

The cell treatment agent of the present invention can be used without being frozen or frozen for later use. If it is to be frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) may be added to the pre-freezing cell population.

In addition, the cell therapy agent may be formulated into a pharmaceutical preparation of a unit dosage form suitable for administration to a patient in accordance with a conventional method in the pharmaceutical field, and the preparation may be administered by one or several doses, . Suitable formulations for this purpose include injections such as ampoules injected as parenteral dosage forms, injecting agents such as injection bags, and spray agents such as aerosol formulations. The injectable ampoule may be mixed with an injection solution immediately before use, and physiological saline, glucose, mannitol, and Ringer's solution may be used as the injection solution. The infusion bag may be made of polyvinyl chloride or polyethylene and may be manufactured by Baxter, Becton Dickinson, Medcep, National Hospital Products or Terumo, An injection bag of a yarn can be exemplified.

In addition to the active ingredient, the pharmaceutical preparation may contain one or more pharmaceutically acceptable inert carriers such as a preservative, an anhydrous agent, a solubilizing agent or a stabilizer in the case of injection, May further comprise a base, an excipient, a lubricant or a preservative.

The cell therapeutic agent or pharmaceutical preparation of the present invention thus prepared may be administered in the form of a mixture with other stem cells used for transplantation and other uses, or a mixture with such stem cells, using administration methods commonly used in the art , Preferably, it is possible to directly engraft or transplant to a diseased part of a patient in need of treatment, or to directly implant or inject into the abdominal cavity, but is not limited thereto. In addition, the above-mentioned administration is both non-surgical administration using a catheter and surgical administration such as implantation or transplant after incision of a disease site, but non-surgical administration method using a catheter is more preferable. In addition to parenteral administration, for example, to direct lesions according to conventional methods, transplantation by intravascular injection, which is a general method of hematopoietic stem cell transplantation, is also possible.

The daily dose of the stem cells may be administered once or several times in the range of 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 . It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the body weight, age and sex of the patient, The scope of the present invention is not limited thereto.

In another embodiment of the present invention, the present invention provides a method of treating a parathyroid gland injury-related disease or osteoporosis, comprising administering to a subject suspected of having hypoparathyroidism or osteoporosis a cell treatment agent comprising the parathyroid gland tissue of the present invention ≪ / RTI >

Administration of the cell treatment agent of the present invention is applicable to any animal. The animal includes human and primate as well as cattle such as cattle, pig, sheep, horse, dog, rat, rat and cat.

As used herein, the term "administering " means introducing the cell therapy agent of the present invention to a patient in any suitable manner, including implantation of differentiated cells. The administration route of the cell therapeutic agent of the present invention can be administered through various routes as long as it can reach the target tissues.

The method of differentiating the parathyroid tissue cells from the one-way stomach cells of the present invention can effectively differentiate the parathyroid tissue cells in a short period of time by using the tonsil tissue to be discarded after the tonsillectomy, and the parathyroid tissue cells obtained from the parathyroid tissue cells, It can produce parathyroid hormone in the same way as tissue cells and regulate the secretion of parathyroid hormone according to the calcium concentration outside the cell. Therefore, it can be effectively used for the treatment of diseases requiring the action of parathyroid hormone including hypoparathyroidism and osteoporosis , And by controlling the extracellular calcium concentration of the parathyroid tissue cells of the present invention, a large amount of parathyroid hormone is secreted out of the cell to easily obtain parathyroid hormone.

FIG. 1 is a diagram illustrating a process of separating one-way stem cells from a tonsil tissue obtained by a tonsillectomy.
Fig. 2 is a diagram showing changes in cell shape from one-way derived cells according to the differentiation period.
FIG. 3 is a view showing the result of the PTH concentration assay in the cell culture medium during the differentiation period. FIG.
FIG. 4 is a graph showing the expression pattern of PTH protein according to the parathyroid gland differentiation period of tonsillar cells. FIG.
Figure 5 is a plot showing the distribution pattern of PTH in the intracellular matrix during differentiation into parathyroid tissue.
6 is a diagram showing intracellular expression and distribution of CHGA, a PTH secretion related protein.
FIG. 7 is a diagram showing the intracellular expression distribution of PTH and CHGA. FIG.
FIG. 8 is a graph showing the regulation of PTH secretion amount according to a change in extracellular calcium concentration. FIG.
FIG. 9 is a diagram showing changes in the cell shape of a somatic stem cell according to a change in extracellular calcium concentration. FIG.
FIG. 10 is a graph showing changes in expression of calcium receptor in mononuclear cells according to changes in extracellular calcium concentration. FIG.
FIG. 11 is a diagram showing the cell microstructure of a mononuclear stem cell differentiated into a parathyroid tissue. FIG.
FIG. 12 is a graph showing the bone forming ability of PTH produced and secreted from a somatic stem cell. FIG.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples.

Example  1: Isolation of somatic stem cells

Ewha Womans University Mokdong Hospital underwent tonsillectomy for 6 patients (5 male, 1 female, mean age 7.2 years) under the age of 10 years. The performance of this study has been reviewed by the Clinical Ethics Committee (ECT 11-53-02). Two thirds of the extracted tonsils were used for clinical histology and the remaining one third was used for this study.

A method for isolating and culturing stem cells from the obtained tonsil tissue is shown in Fig.

Specifically, the collected tonsil tissues were washed with physiological saline and then treated with collagenase type I (Invitrogen) and 10 μg / ml DNAse (Sigma-Aldrich, St. Louis, MO ) Were pulverized in a tissue culture medium RPMI-1640 (Roswell Park Memorial Institute medium 1640, Invitrogen Corporation, Carlsbad, Calif.) At 37 ° C for 30 minutes. The suspended tissues were centrifuged and filtered to obtain cells. The obtained cells were washed twice with RPMI 1640/20% normal human serum (PAA Laboratories, GmbH, Paching, Austria) and once with RPMI 1640/10% NHS. The washed cells were subjected to gradient centrifugation in Ficoll-Paque (GE Healthcare, Little Chalfont, Buckinghamshire, UK) to obtain mononuclear cells. The mononuclear cells 10 ⁸ 10% fetal calf serum (Fetal bovine serum, FBS, Invitrogen ), 100㎍ / ㎖ streptomycin and 100U / ㎖ ampicillin DMEM-HG (Dulbecco's modified Eagle 's medium-High Glucose) containing (Invitrogen) And cultured for 48 hours. After incubation, unattached cells were removed and adherent cells considered to be tonsillar mesenchymal stem cells (T-MSC) were subcultured for 3-5 generations over 4 weeks It was used in this experiment.

Example  2: induction of differentiation into parathyroid tissue

In order to confirm whether T-MSC can be differentiated into endodermally differentiated cells, T-MSC was induced to differentiate into parathyroid cell-like cells which are endodermal cells. Specifically, T-MSC was cultured in 5% FBS, The cells were inoculated into DMEM medium containing actin A (100 ng / ml, R & D System, Inc. Minneapolis) and sonic hedgehog (Shh, 100 ng / ml, R & D Systems) For 21-25 days.

As a result of culturing, it was observed that as the differentiation period elapsed, the stromal stem cells formed a multi-layer accumulating in several layers as shown in FIG. 2 and formed a cubic cell shape.

Example  3: PTH of Degree of secretion  Research

When the cell culture medium containing the differentiation inducer is replaced by the differentiation period (0, 4, 7, 10, 15, 20 days), the existing cell culture fluid is collected and the PTH secreted outside the cell by the differentiation process into the parathyroid tissue Was used as a sample for analysis. The collected cell culture was filtered with a syringe filter having a size of 0.45 μm to remove cell suspension and cell debris. The filtered cell culture was lyophilized and concentrated using a freeze dryer (Operon).

The lyophilized powder form was resuspended in phosphate buffered saline (pH 7.4) and the PTH concentration was analyzed by immunochemical method. That is, the PTH concentration excreted by the ELCIA method using the binding force of the antibody to the PTH protein was examined.

As a result, as shown in FIG. 3A, in the cells treated with the differentiation inducing solution, a statistically significant amount of PTH was secreted into the cell culture fluid as compared with the non-differentiated cells. The concentration of PTH in the range of 15-60 pg / ml, which is the normal PTH concentration in vivo, was increased up to 40 pg / ml. (FIG. 3B). As a result of the cumulative PTH concentration value, cumulative PTH secretion amount after 10 days from the treatment of the differentiation inducing solution was irradiated at a concentration of 100 pg / ml. When the differentiated stromal cells were continuously cultured, 100 pg / ml (Fig. 3C). ≪ tb >< TABLE >

Example  4: Western Blot  By method PTH  Investigation of Protein and Related Protein Expression

Cells were washed twice with PBS (phosphate buffered saline, pH 7.4) and then stained with RIPA buffer (25 mM Tris-Cl pH 7.6, 150 mM NaCl, 1% NP- 40, 1% sodium deoxycholate, 0.1% SDS). The extracted protein was quantitated by BCA (bicinchoninic acid) method using albumin as a standard solution and used for the investigation of intracellular PTH protein expression.

10 to 12% of SDS-polyacrylamide gel (SDS-PAGE) was prepared, and 30 μg of the protein sample quantified for each step of differentiation period was loaded on SDS-PAGE and subjected to electrophoresis, My proteins were separated by size. After electrophoresis, proteins of various sizes separated on the gel were transferred to a nitrocellulose membrane by an electrical method.

(Ab frontier, LF-MA0140), a calcium sensing receptor (CaSR, Abcam ab18200), and a PTH-specific antibody were added to the membrane taken out of the membranes corresponding to the PTH protein size among the proteins of each size transferred onto the membrane. , Cystein rich protein 61 (Cyr61, Santa Cruz SC-13100) and osteocalcin (OC, Santa Cruz SC-74495), chromagranin A (CHGA, AVIVA ARP-41930- ) Was added and reacted with the antibody at a low temperature of 4 ° C for 14-16 hours. After the reaction with the primary antibody (diluted 1: 1000), mouse-IgG-HRP (horse radish peroxidase) conjugate, a secondary antibody specific to the primary antibody, was further reacted (diluted at a ratio of 1: 3000). The extent of binding of PTH antibody bound to intracellular PTH protein was investigated by ECL (enhanced chemiluminescence) method. In other words, the degree of expression of PTH, CaSR, CHGA, and Cyr61 in the extracted proteins was examined by detecting the degree of ECL reaction by the HRP enzyme in the secondary antibody bound to the primary antibody, PTH antibody.

At the same time, the same amount of protein was used for each sample. To verify that the entire Western blotting method was consistently performed, the degree of expression using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) Respectively.

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

As a result, as shown in Fig. 4, the expression of PTH protein in tonsil cells was increased as the differentiation period elapsed. Especially, 4 days after induction of differentiation, it was significantly increased and maintained until 7 days.

Example  5: Confocal  Using a microscope PTH  And PTH  Investigation of secretory granule protein distribution

Intracellular distribution of proteins related to PTH and PTH secretion regulation in T-MSC cells during the process of differentiating into parathyroid tissue was examined using confocal laser scanning microscope (LSM-5 Pascal EXCITER, Carl Zeiss) .

In order to treat the cells to be used for confocal microscopy, non-differentiated cells and differentiated cells were prepared at various differentiation periods (7, 14, and 21 days) and cover-glass was prepared in advance on each well bottom - < / RTI > 10 ⁴ < / RTI >

Cells of each differentiation period were washed with PBS and the cells were fixed with 10% formalin solution. Immobilized cells were treated with 2% BSA (bovine serum albumin) solution to inactivate nonspecific proteins, followed by treatment with 2% BSA solution containing primary antibody (PTH, CHGA, Cyr61) Lt; / RTI > After the reaction with the primary antibody, the unreacted antibody was washed twice with PBS and then incubated with a secondary antibody (mouse IgG-FITC, rabbit-IgG-Rhodamine, Invitrogen) 2% BSA solution was reacted at 37 占 폚 for 1 hour. At this time, DAPI (4 ', 6-Diamidino-2-Phenylindole, Dihydrochloride) (Molecular Probes®), which is specifically stained with nuclei, was added to simultaneously stain the nuclei in each cell. After completion of the reaction with the secondary antibody, the cell surface was washed with PBS twice, the cover glass with the cells attached thereto was removed, placed on a glass for fluorescence microscopy, adhered to the glass with mounting solution, The expression of each of the antibodies on a microscope and the distribution pattern thereof were examined.

As a result, as shown in FIG. 5, during the total differentiation period, the expression of intracellular PTH showed a large change in the PTH distribution in the cell group (control cell, denoted by C) It was investigated not to appear. On the other hand, in the case of treatment with differentiation inducer (differentiated cell, denoted as D), the expression of PTH was remarkably increased as the differentiation period elapsed. This pattern showed a markedly higher expression distribution on day 14, showing the same tendency as the expression patterns of PTH protein in the cells and the changes in the secreted PTH concentration.

In addition, the expression pattern of PTH dispersed in the cytoplasm is expressed in the form of granules, and the form of the produced PTH is different from that of other secretory hormones such as secretory granule formation and similar mechanisms, It could be predicted.

Regarding the distribution of PTH in the cells, the intracellular distribution pattern of CHGA was examined. As a result, it was found that the expression was not observed in unilocated tonsil cells as shown in FIG. 6. However, in the differentiated tonsil cells, The expression of CHGA was increased and its degree was evenly distributed in cytoplasm.

PTH and CHGA were double-stained at the same time, and the distribution pattern of PTH and CHGA was found to be coincident with that of PTH and CHGA, and it was found that CHGA was directly related to the storage and secretion of the produced PTH 7).

Example  6: Depending on the extracellular calcium concentration PTH Secretion control ability  Research

PTH is a major hormone that acts to maintain the homeostasis of calcium in the body by recognizing changes in the extracellular calcium concentration. In vivo application and use of PTH produced in parathyroid tissue cells differentiated from tonsil cells, it is important to investigate whether PTH normally functions PTH-specific.

Therefore, we investigated the changes of PTH secretion according to the changes of external calcium concentration by differentiating extracellular calcium concentration after differentiating tonsillar cells into parathyroid tissue.

First, T-MSC was treated with differentiation inducer for 7 days to induce differentiation into parathyroid tissue. After induction of differentiation for 7 days, the cells differentiated into the parathyroid gland were cultured in the culture medium in which the calcium concentration in the cell culture medium was changed to the normal concentration (1.5 mM), low concentration (0.09 mM) and high concentration (3.0 mM). The T-MSC cultured in the culture medium containing various calcium concentrations was exposed for 3, 8, 24, and 48 hours, and the cell culture medium was collected at each time point. The amount of PTH secreted into the cell culture medium according to the external calcium concentration was measured in the same manner as in Example 3 ELCIA method.

As a result, as shown in FIG. 8, the secretion amount of PTH was shown to be higher than that of normal calcium concentration in the low calcium state, especially after 24 hours exposure. On the other hand, the amount of PTH secreted in the high calcium state was significantly lower than that in the normal calcium concentration state. It is possible that the differentiated parathyroid tissue can also regulate the degree of secretion of PTH according to the extracellular calcium concentration and control the absorption / reabsorption of calcium through regulation of secretion of PTH to perform a unique function of PTH .

Particularly, in the low calcium state, the cell shape of the tonsil cell also shows that many granule-shaped particles are formed, and it is also shown that the cell shape change due to secretory granule formation can also be induced (Fig. 9).

In addition, a calcium receptor (CaSR) is known to recognize the extracellular calcium concentration and regulate the secretion of stored PTH. CaSR is a large number of receptors on the surface of parathyroid cells. It plays a role in maintaining and regulating the homeostasis of calcium concentration in the blood while recognizing the calcium concentration outside the cells. (PTH) receptor (PTHR), which is present on the surface of osteoblasts, is transferred to the bone tissue through blood circulation, And the calcium is released into the blood while reacting, so that the calcium homeostasis is maintained in vivo. It is known that the activity of CaSR is controlled by the amount of the receptor itself. We investigated the expression level of CaSR and the level of PTH expression by using endothelial cells exposed to various extracellular calcium concentrations and investigated the relationship between CaSR expression and the action of PTH on CaSR production.

As a result, as shown in Fig. 10, the expression level of CaSR increases in the low calcium state, and the expression level decreases in the high calcium state, and CaSR plays a key role in the normal calcium concentration homeostatic regulation of PTH produced in the tonsillar cells .

Example  7: Stem cells differentiated into parathyroid tissue Intracellular  Confirm microstructure

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 functions of specific organs.

The intracellular microstructure of mononuclear stem cells differentiated into parathyroid tissue was examined by electron microscopy to investigate similarity with normal parathyroid tissue.

In order to investigate the changes in intracellular microstructure of T-MSCs differentiated into parathyroid glands, they were observed using an electron microscope. The changes in intracellular microstructure were investigated using the cells at the 7th day of differentiation, which is the maximum PTH secretion ability. At this time, Actin A, which is a differentiation inducing agent, and cells on day 7 not treated with Shh were used as a control group. The control cells (Control, C) and differentiation (D) cells were washed with PBS and treated with trypsin to remove adhered cells. After centrifugation at 1200 rpm, The cells were collected by centrifugation for a minute. Cells collected in pellet form were stored in 2.5% glutaraldehyde solution and fixed at 4 ° C.

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

When unilateral one-way stem cells and parathyroid-differentiated one-way stem cells were compared and observed, it was found that the differentiated unilateral one-way stem cells exhibit the basic structure of nucleus, mitochondria, and Golgi, similar to other normal cells ).

On the other hand, in the cells induced to differentiate into parathyroid glands, a number of structures considered to be cell granules were formed in addition to the basic cell microstructure (FIG. 11B). In addition, a structure similar to that of a pro-secretory granule was also observed (FIG. 11B), and it was structurally confirmed that the differentiation into a hormone-secreting tissue that secretes PTH proceeded well.

Example  8: Osteoclasts  Used Osteotomy  Research

Since PTH has been reported to have bone morphology through the PTH receptor (parathyroid hormone receptor, PTHR) on the osteoblast surface, the role of PTH as a therapeutic agent for new osteoporosis has been suggested through induction of direct bone function of PTH itself.

Based on this, we investigated whether PTH produced and secreted from one-way stem cells differentiated into parathyroid tissue actually had PTH-specific bone formation capability.

We investigated the osteoarthritic function of cell culture obtained from T-MSCs differentiated into PTH-releasing parathyroid tissue using preosteoblast MC3T3-E1 cells. That is, the MC3T3-E1 cells were divided into 10 ⁴ cells per well on a 24-well plate, and a bone cell culture solution (Invtorgen) containing ascorbic acid 2-phosphate, dexamethasone and? -Glycerophosphate was treated for 2 weeks The osteotomy function was investigated. The culture medium was changed every 3-4 days and the conditioned medium (CM) containing PTH secreted outside the cell was treated at various concentrations (10, 50, 100, 1000 pg / ml), and bone formation ability of secreted PTH was examined. The 17-beta estradiol (10 μM) used in clinical trials was compared with a positive control drug by hormone therapy of PTH and osteoporosis synthesized and commercialized by recombinant methods.

In order to investigate the bone morphology, we used the Alizarin red S method to stain cytoplasmic expression of osteocalcin (OC) protein and accumulation of calcium in the cytoplasm, which are representative markers of bone formation by Western blotting.

That is, after the incubation was completed, the culture solution was removed, and the cells attached to the bottom of each well were washed with PBS, fixed with 60% isopropyl alcohol for 5 minutes, washed twice with distilled water, % Alizarin red S aqueous solution (pH 4.2) for 3 minutes. After completion of the staining, the Alizarin red S aqueous solution was removed, immediately washed with distilled water three times, and then observed with a phase contrast microscope and compared with the control (MC3T3-E1 cultured without inducing differentiation)

The expression of osteocalcin protein, a typical osteocalcin marker, was increased and the expression of osteocalcin was also increased with increasing concentration of CM. The degree of osteocalcin expression was lower than that of hormone estrogen, (Fig. 12). As shown in Fig. In addition, when CM (1000 pg / ml = 1 ng / ml) corresponding to 1/100 of the commercial PTH concentration was treated, compared with the case where the commercial PTH (104 ng / ml) Similar bone morphology was demonstrated and it was confirmed that PTH produced and secreted from the somatic stem cells possessed high physiological activity.

It was also confirmed that CM-induced osteogenesis was induced by calcification of intracellular calcified parts using Alizarin red-S staining, and calcified and stained parts were significantly increased.

The results show that the PTH produced by the method of the present invention can perform physiological functions inherent to PTH when applied in vivo and can be used as a therapeutic agent.

All data were expressed as mean ± standard deviation (SD), and statistical significance was analyzed by Student's t-test using GRAPHPAD PRISM software (GraphPad Software Inc., San Diego, Calif.). In all analyzes, P <0.05 was considered statistically significant. All experiments were repeated at least 3 times.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (19)

A method of differentiating parathyroid tissue cells from a somatic stem cell, comprising culturing the somatic stem cells in a medium comprising Activin A and Sonic hedgehog (Shh).
3. The method of claim 1, wherein the actin A and the sonic hedgehog are each present in the medium at a concentration of 50 ng / ml to 300 ng / ml.
3. The method of claim 2, wherein the actin A and the sonic hedgehog are each present in the medium at a concentration of 100 ng / ml.
The method of claim 1, wherein the medium is Dulbecco's modified Eagle medium.
The method of claim 1, wherein the incubation is performed for at least 4 days.
The method according to claim 1, wherein the one-way stem cells are mesenchymal stem cells.
The method according to claim 1, further comprising the step of replacing fresh medium every 4 to 5 days when the culture is performed for 4 days or more.
i) culturing the somatic stem cells in a medium containing Activin A and Sonic hedgehog (Shh) to differentiate into parathyroid tissue cells and ii) culturing the cultured solution or cell lysate obtained in step i) And separating the parathyroid hormone from the liquid. &Lt; RTI ID = 0.0 &gt; 1. &lt; / RTI &gt;
9. The method of claim 8, further comprising, after step i), regulating the calcium concentration of the culture medium to secrete the parathyroid hormone out of the cell.
10. The method of claim 9, further comprising, after step i), adjusting the calcium concentration of the culture to below 0.5 mM.
9. The method of claim 8, wherein the actin A and the sonic hedgehog are each present in the medium at a concentration of 50 ng / ml to 300 ng / ml.
9. The method of claim 8, wherein the actin A and the sonic hedgehog are each present in the medium at a concentration of 100 ng / ml.
9. The method of claim 8, wherein the incubation is performed for more than 4 days.
9. The method of claim 8, wherein the incubation is performed for at least 7 days.
Parathyroid tissue cells differentiated from one - way stem cells.
16. A parathyroid tissue cell according to claim 15, which is differentiated by the method of any one of claims 1 to 7.
16. The parathyroid tissue cell according to claim 15, which produces a parathyroid hormone.
A cell therapy agent for the treatment of hypoparathyroidism or osteoporosis comprising parathyroid tissue cells differentiated from one-way stem cells.
A method for treating hypoparathyroidism or osteoporosis comprising administering to a non-human subject suspected of having hypoparathyroidism or osteoporosis a cell therapy agent comprising parathyroid tissue cells differentiated from mononuclear stem cells.

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