KR20230111762A - A use of PCOLCE2 for predicting leukocyte function promoting of Tonsil-derived mesenchymal stem cell or culture medium thereof - Google Patents

Abstract

The present invention provides the use of PCOLCE2 for predicting enhancement of leukocyte function of tonsillar-derived mesenchymal stem cells or their culture medium.
PCOLCE2 according to the present invention provides information on the effect of tonsillar-derived mesenchymal stem cells or their culture on enhancement of white blood cell function to solve the problem of heterogeneity according to the combination of various precursor cells. Through this, it is possible to determine tonsillar-derived mesenchymal stem cells or their culture medium suitable for administration as a combination or adjuvant to bone marrow transplantation or chemotherapy to enhance immunity, and by using this, it can be used for the development of therapeutic agents that can be applied to patients with a reduced number of white blood cells, such as a state in which immunity is reduced after bone marrow transplantation or chemotherapy.

Description

Use of PCOLCE2 for predicting leukocyte function promotion of tonsillar-derived mesenchymal stem cells or their culture medium

The present invention relates to a composition for predicting enhancement of leukocyte function in tonsillar-derived mesenchymal stem cells or their culture medium, including a preparation for measuring the expression level of mRNA or protein of PCOLCE2, a kit containing the composition, a method for screening tonsillar-derived mesenchymal stem cells or a culture medium for enhancing white blood cell function using a preparation for measuring the expression level of PCOLCE2 mRNA or protein, and a method for screening tonsillar-derived mesenchymal stem cells selected by the screening method or a culture medium thereof, and a gene or protein of PCOLCE2 It relates to a pharmaceutical composition for preventing or treating leukopenia comprising any one selected.

As the first line of innate defense and effectors of adaptive immunity, neutrophils participate in immune-mediated defense against invading pathogens. Neutrophils trap and kill microorganisms using dynamic strategies such as phagocytosis, neutrophil extracellular trap generation, granule secretion, and reactive oxygen species (ROS) formation. ROS production, also known as oxidative burst, is particularly essential for neutrophils to destroy and eliminate invading microorganisms, and defects in ROS production lead to bacterial survival and uncontrolled infection.

Serious infectious diseases due to neutrophil dysfunction are an important cause of morbidity and mortality worldwide, such as during chemotherapy-induced neutropenia, fatal infections are known to be one of the leading causes of chemotherapy-related deaths. In addition, infections are known to be the main cause of death occurring within 30 days after allogeneic hematopoietic stem cell transplantation, and defective innate immune responses, including neutrophil dysfunction, have been reported to be host susceptibility factors during bacterial infections in diabetes. That is, since infection is significantly associated with morbidity and mortality in patients receiving allogeneic neutrophil cells, enhancing neutrophil function is critical to maintaining host immune defenses against invading microorganisms during bone marrow transplantation. Thus, enhancement of neutrophil function, including optimal ROS formation, may be useful in the prevention of serious infections associated with morbidity and mortality.

On the other hand, mesenchymal stem cells (MSCs) are non-hematopoietic, pluripotent fibroblast-like cells that exhibit the efficacy of differentiating into mesodermal lineages including osteocytes, adipocytes and chondrocytes. MSCs can be easily isolated from various tissues, including bone marrow, adipose tissue, amniotic fluid, and Wharton's jelly (substantia gelatinea funiculi umbilicalis), and have immunomodulatory capabilities, enabling successful allogeneic transplantation. In addition, mesenchymal stem cells can be derived from various types of tissues. However, it is known that mesenchymal stem cells from various sources can have heterogeneity depending on differences in culturing methods in in vitro growth culture conditions, differences in stem cell donor sources, differences in stem cell extraction sites, and the like. As a result of these problems, the stability problem of stem cells is continuously emerging, and research and development on a new method for collecting stem cell sources having homogeneous characteristics is required.

On the other hand, tonsil-derived mesenchymal stem cells (TMSCs) are superior to other MSCs in terms of proliferation rate, differentiation efficacy, and immunomodulatory ability. Because TMSCs successfully retain MSC-specific properties during freezing and thawing and up to passage 15 during long-term in vitro cell culture, TMSCs are emerging as good MSC candidates for clinical cell therapy.

Under the above background, the present inventors have tried to find a method for obtaining tonsillar-derived mesenchymal stem cells or their culture medium, which can be effective in reducing immunity caused by bone marrow transplantation or chemotherapy. In the process, it was confirmed that tonsillar-derived stem cells exhibit diverse characteristics depending on the donor, and the present invention was completed by confirming that tonsillar-derived stem cells with high PCOLCE2 expression and their culture medium can excellently support leukocyte function, particularly the ROS production ability of neutrophils.

Liu et al. J Transl Med (2019) Transfus Med Hemother 2008;35:272-277 J Immunol 2009; 183:993-1004 Batsali et al. Stem Cell Research & Therapy (2017) 8:102

One object of the present invention is to provide a composition and/or kit for predicting enhancement of leukocyte function in amygdala-derived mesenchymal stem cells or their culture medium, including an agent for measuring the expression level of PCOLCE2 mRNA or protein.

Another object of the present invention is to provide a composition and/or kit for screening tonsillar-derived mesenchymal stem cells or their culture medium for enhancing immunity, including an agent for measuring the expression level of mRNA or protein of PCOLCE2.

Another object of the present invention is to provide a method for screening tonsillar-derived mesenchymal stem cells or their culture medium that enhances leukocyte function using an agent for measuring the expression level of PCOLCE2 mRNA or protein.

Another object of the present invention is to provide a method for screening tonsillar-derived mesenchymal stem cells or a culture medium thereof for enhancing immunity using an agent for measuring the expression level of PCOLCE2 mRNA or protein.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating leukopenia comprising tonsil-derived mesenchymal stem cells selected by the above screening method or their culture medium, any one selected from the gene or protein of PCOLCE2 and leukocyte cells.

As one aspect for achieving the above object, a composition for predicting enhancement of leukocyte function in amygdala-derived mesenchymal stem cells or their culture medium containing an agent for measuring the expression level of PCOLCE2 mRNA or protein is provided.

Preferably, a composition for predicting enhancement of leukocyte function of tonsillar-derived mesenchymal stem cells or their culture medium containing an agent for measuring the expression level of PCOLCE2 mRNA or protein is provided.

"PCOLCE2 (Procollagen C-Endopeptidase Enhancer 2)" is an extracellular matrix glycoprotein that serves as a functional procollagen C-proteinase enhancer, binds to the COOH-terminal propeptide of type I procollagen and functions to enhance the cleavage of procollagen by procollagen C-proteinases such as BMP (Bone morphogenetic protein) 1 (Gene ID: 26577).

In the present invention, the term "mesenchymal stem cell" refers to a cell having infinite proliferation ability (self-renewal capacity) in an undifferentiated state and the ability (multi-differentiation potency) to be differentiated into cells of various tissues by a specific differentiation inducing stimulus. Mesenchymal stem cells may be combined into various precursor cells according to their extraction site and have heterogeneity in cell composition. Since the diversity of these cells can lead to variations in non-clinical and clinical tests, etc., there is a need to classify mesenchymal stem cells having homogeneous characteristics.

In the present invention, the term "tonsil" refers to a tissue located in the back of the neck and the back of the nose that primarily protects the body from substances such as bacteria invading from the outside and at the same time acts as a lymphatic epithelial immune tissue. The tonsils include adenoids, adenopharyngeal tonsils, palatine tonsils, lingual tonsils, and the like. In the present invention, the amygdala is used as a source tissue for providing amygdala-derived mesenchymal stem cells.

The term "tonsil derived mesenchymal stem cells" of the present invention refers to mesenchymal stem cells (MSCs) derived from tonsillar. The tonsil-derived mesenchymal stem cells may be of human origin. Tonsil-derived mesenchymal stem cells are considered good candidates for clinical cell therapy because they are superior to other mesenchymal stem cells in terms of proliferation rate, differentiation potency, and immune modulatory ability, successfully maintain specific properties of mesenchymal stem cells during freezing and thawing, and maintain up to passage 15 during long-term in vitro cell culture.

The culture period of tonsillar-derived mesenchymal stem cells is preferably 4 days or more, more preferably 7 days or more, and even more preferably 7 to 30 days or less. Preferably, the tonsil-derived mesenchymal stem cells may be collected and used from tonsil tissue isolated from a patient who has undergone tonsillectomy, but is not limited thereto. When collected from tonsil tissue isolated from a patient, the collected tonsil tissue is washed with a buffer, treated with an enzyme mixture to disintegrate connective tissue, at this time, the suspended tissue is centrifuged, and the cell mixture in the cut tissue is cultured using cell fractionation and centrifugation using Ficoll to only the precipitated portion of the lower layer, isolating only the cell portion showing the ability to adhere to the cell bottom, and then immunochemical using a cell surface marker among cells showing the ability to adhere to the bottom of the culture dish. Tonsil-derived mesenchymal stem cells can be isolated using the method.

As used herein, the term "Tonsil-derived mesenchymal stem cell culture medium (T-MSC CM)" refers to a medium in which tonsil-derived mesenchymal stem cells are cultured and only the culture medium is collected. In the present invention, the culture solution of tonsil-derived mesenchymal stem cells may be a culture solution obtained after culturing tonsil-derived mesenchymal stem cells and removing the cells, a culture supernatant, a concentrate thereof, or a dried product thereof.

"Tonsil-derived mesenchymal stem cell culture medium" of the present invention is a medium containing growth factors, cytokines, etc. extracted from tonsil-derived mesenchymal stem cells in culture, and the composition of active ingredients is different from that of other-derived mesenchymal stem cell cultures according to the expression characteristics of the tonsil-derived mesenchymal stem cells. In the present invention, the method for obtaining a culture medium of tonsillar-derived mesenchymal stem cells can be obtained by various methods known in the art. For example, in order to obtain a culture medium of tonsillar-derived mesenchymal stem cells, the tonsil-derived mesenchymal stem cells are cultured until about 60-90% confluency, and then the culture medium is replaced with a new medium. Then, after culturing the cells further, the culture medium is collected, concentrated, and used.

In the present invention, the composition of the present invention classifies excellent tonsillar-derived mesenchymal stem cells or their culture medium that enhances leukocyte function, in particular, the ROS-producing ability of neutrophil cells, with the above homogeneous characteristics, thereby enhancing immunity, assisting in bone marrow transplantation, as well as infection-related morbidity and mortality.

In addition, even tonsillar-derived mesenchymal stem cells extracted from the same extraction site in the present invention may have different characteristics depending on the donor providing them, so that excellent tonsillar-derived mesenchymal stem cells or their culture are classified among tonsil-derived mesenchymal stem cells derived from the same source tissue to provide cells or their culture suitable for patients in need of immune enhancement, bone marrow transplantation or chemotherapy.

When neutrophil cells and tonsil-derived mesenchymal stem cells or their culture are administered together, the ROS-producing ability of neutrophil cells can be promoted. Since infections are significantly associated with morbidity and mortality in patients receiving allogeneic neutrophil cells, enhancing neutrophil function is important for maintaining host immune defenses against invading microorganisms during bone marrow transplantation or chemotherapy. When the tonsil-derived mesenchymal stem cells or their culture medium having high expression of PCOLCE2 mRNA or protein according to the present invention are administered together with neutrophil cells, the reactive oxygen species (ROS) generating ability of the neutrophil cells is greatly enhanced, thereby greatly enhancing the efficacy of host immune defense in transplanted individuals.

In the present invention, "prediction of enhancement of leukocyte function of tonsillar-derived mesenchymal stem cells or their culture medium" is to provide information on whether or not the tonsillar-derived mesenchymal stem cells or their culture medium that enhances the leukocyte function, in particular, the ROS generating ability of neutrophil cells administered for therapeutic purposes. Specifically, it is for selection/classification that can provide information to confirm that tonsillar-derived mesenchymal stem cells or their cultures show differences in the ROS generating ability of neutrophil cells. For the purpose of the present invention, it means that it is possible to detect whether they are tonsillar-derived mesenchymal stem cells or their culture medium capable of increasing the ROS-producing ability of neutrophil cells due to the high expression level of PCOLCE2, or stem cells or their culture medium with low availability, which have a low level of expression of the gene and the effect of improving the ROS-generating ability of neutrophil cells.

According to one embodiment of the present invention, when tonsillar-derived mesenchymal stem cells or their culture medium expressing high PCOLCE2 are treated together with leukocytes (neutrophil cells), leukocyte function, in particular, the ROS generating ability of neutrophil cells is greatly increased. Accordingly, it is highly likely to be used as a therapeutic agent/adjuvant for bone marrow transplantation or chemotherapy. On the other hand, when tonsillar-derived mesenchymal stem cells with low PCOLCE2 expression or their culture medium are treated with leukocytes (neutrophil cells), the improvement effect in terms of leukocyte function, particularly ROS generating ability of neutrophil cells, is very low. Accordingly, in the case of these cells or their culture medium, they can be classified as cells or culture medium not used for bone marrow transplantation or chemotherapy. Through this information, tonsillar-derived mesenchymal stem cells or their culture medium suitable for bone marrow transplantation or chemotherapy can be selected and classified using the composition according to the present invention.

Therefore, in the present invention, information can be provided to select tonsillar-derived mesenchymal stem cells or their culture medium for immunity enhancement by using an agent for measuring the expression level of the mRNA or protein of gastric PCOLCE2.

As described above, the present invention, as one aspect for achieving the above object, provides a composition for screening tonsillar-derived mesenchymal stem cells or their culture medium for enhancing immunity, including an agent for measuring the expression level of mRNA or protein of PCOLCE2.

In the present invention, immunity enhancement, for example, when an immune response that is not beneficial to the living body induced by an external antigen due to invasion of microorganisms occurs, for example, an immune defense ability caused by an immune response induced by an external antigen. It can mean an improvement effect. In addition, it may exhibit a more improved action in achieving an appropriate balance between the risk of opportunistic infection due to immunosuppression and immunosuppression that occurs in some cases.

The composition provides information on tonsillar-derived mesenchymal stem cells or their culture medium that enhances the function of leukocytes, in particular, the ROS generating ability of neutrophil cells. That is, the selected tonsillar-derived mesenchymal stem cells or their culture greatly increase the function of leukocytes, particularly the ability of neutrophil cells to generate ROS.

Agents for measuring the level of mRNA in the technical field of the present invention are preferably primers or probes, but are not limited thereto.

A primer is a nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairing with a complementary template and refers to a short nucleic acid sequence that functions as a starting point for template strand copying.

A probe refers to a natural or modified nucleic acid fragment such as RNA or DNA capable of hybridizing to an mRNA and a specific nucleotide sequence and being labeled so that the presence or absence of a specific mRNA can be confirmed. Probes used for hybridization may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like. Conditions suitable for hybridization can be determined by adjusting the temperature, ionic strength (buffer concentration), and the presence of compounds such as organic solvents. These stringent conditions may be determined differently depending on the sequences to be hybridized.

A person skilled in the art can design a primer pair that specifically amplifies a specific region of the gene or a probe that specifically recognizes a specific region of the gene from the known nucleic acid sequence of the gene of the present invention, and chemically synthesized using a method known in the art. In addition, it may be modified using a label such as a radioactive isotope, a fluorescent molecule, or biotin to directly or indirectly provide a detectable signal.

Agents for measuring protein levels in the present art are preferably antibodies, and include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies each specifically binding to PCOLCE2. Antibodies of the present invention also include complete forms having two full-length light chains and two full-length heavy chains and functional fragments of antibody molecules, such as Fab, F(ab'), F(ab') 2 and Fv forms.

Each antibody specific to PCOLCE2 includes both existing antibodies as well as antibodies that can be prepared by known methods.

Using this, immunoassays (e.g., radioimmunoassay method, radioactive immunoprecipitation method, enzyme-linked immunosorbent method (ELISA), dot blot analysis, western blot, inhibition or competition assay, and sandwich assay) can be quantitatively or qualitatively performed.

As one aspect for achieving the above object, to provide a kit comprising a composition for predicting enhancement of leukocyte function of tonsillar-derived mesenchymal stem cells or their culture medium containing an agent for measuring the expression level of mRNA or protein of PCOLCE2.

In the present invention, bone marrow transplantation or chemotherapy adjuvant refers to an agent that can be used as an adjuvant to treat or prevent immune decline that occurs after bone marrow transplantation or chemotherapy commonly used in the art. By using the adjuvant according to the present invention, bone marrow transplantation or chemotherapy can be promoted by promoting neutrophil ROS generation ability in a subject requiring chemotherapy, thereby enhancing the effect of bone marrow cells or a therapeutic agent containing the same. Tonsil-derived mesenchymal stem cells or a culture medium thereof may be used as such an adjuvant or a role of an adjuvant.

As one aspect for achieving the above object, to provide a kit comprising a composition for screening tonsillar-derived mesenchymal stem cells or their culture medium for enhancing immunity, including an agent for measuring the expression level of mRNA or protein of PCOLCE2.

The kit includes primers or antibodies capable of selectively recognizing genes related to increased or decreased expression of PCOLCE2 or proteins thereof from tonsillar-derived mesenchymal stem cells or their culture medium, as well as tools and reagents commonly used in the art used for immunological analysis. Such tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminating agent. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, polyacrylonitrile, fluororesins, cross-linked dextrans, polysaccharides, polymers such as latex-plated magnetic particulates, other papers, glass, metals, agarose, and combinations thereof.

The kit may have the form of a reverse transcription polymerase chain reaction (RT-PCR) device, a real time polymerase chain reaction (Real time PCR) device, an ELISA plate, a dip-stick device, an immunochromatography test strip and a radiation partitioning immunoassay device, and a flow-through device, and is preferably composed of a microarray, but is not limited thereto. In addition, when antibodies are provided on a protein chip on which a plurality of proteins can be immobilized, the formation of antigen-antibody complexes for two or more antibodies can be observed.

As one aspect for achieving the above object, a method for screening tonsillar-derived mesenchymal stem cells or a culture thereof that enhances leukocyte function using an agent for measuring the expression level of PCOLCE2 mRNA or protein is provided.

As one aspect for achieving the above object, a method for screening tonsil-derived mesenchymal stem cells or a culture medium thereof for enhancing immunity using an agent for measuring the expression level of PCOLCE2 mRNA or protein is provided.

In a specific embodiment, a method for screening tonsillar-derived mesenchymal stem cells or a culture thereof that enhances leukocyte function using an agent for measuring the expression level of PCOLCE2 mRNA or protein includes: (a) tonsillar-derived mesenchymal stem cells or obtaining a sample from a culture thereof; (b) measuring the expression level of PCOLCE2 mRNA or protein in the sample; and (c) selecting tonsillar-derived mesenchymal stem cells or their culture medium in which the expression level of PCOLCE2 in step (b) is increased compared to the reference value. This method can be done in vitro.

In addition, in a specific embodiment, a method for screening tonsil-derived mesenchymal stem cells or a culture thereof for enhancing immunity using an agent for measuring the expression level of mRNA or protein of PCOLCE2 includes: (a) tonsillar-derived mesenchymal stem cells or obtaining a sample from a culture thereof; (b) measuring the expression level of PCOLCE2 mRNA or protein in the sample; and (c) selecting the tonsil-derived mesenchymal stem cells or their culture medium in which the expression level of PCOLCE2 in step (b) is increased compared to the reference value as tonsil-derived mesenchymal stem cells or their culture medium for immunity enhancement. This method can be done in vitro.

Specifically, the step of obtaining a sample of step (a) is a step of obtaining a normal mRNA or protein sample for measuring the expression level of mRNA or protein. Such a sample can be easily obtained from tonsillar-derived mesenchymal stem cells themselves or their culture medium using a method known in the art or a kit for obtaining a sample.

In step (b), the mRNA or protein expression level of PCOLCE2 can be measured using the conventional method for measuring the mRNA or protein expression level mentioned above. For example, a reverse transcription polymerase chain reaction (RT-PCR) device, a real time polymerase chain reaction (Real time PCR) device, an ELISA plate, a dip-stick device, an immunochromatography test strip and a radiation partitioning immunoassay device, and a flow-through device, etc. The expression level of PCOLCE2 can be measured through the above-mentioned content and a measurement method within the scope of a conventional technique using a flow-through device.

From the above results, it is possible to confirm the expression level of PCOLCE2, which increases or decreases differently depending on the type of tonsil-derived mesenchymal stem cells or their culture (ie, tonsil-derived mesenchymal stem cells obtained from different donors or donors, or their culture). Based on the results, it is possible to select tonsillar-derived mesenchymal stem cells or their culture, or to screen tonsil-derived mesenchymal stem cells or their culture for enhancing immunity.

Here, the reference value or reference level may be the expression level of PCOLCE2 expressed in conventional stem cells. Alternatively, it may be the expression level of PCOLCE2 measured in stem cells or their culture medium having a low level of ROS generating ability of neutrophil cells. Although this reference value or reference level may vary slightly depending on the purpose, it clearly provides an appropriate reference level for classifying cells suitable for use with neutrophil cells administered in vivo.

The present invention also provides tonsil-derived mesenchymal stem cells or a culture thereof, selected according to a method for screening tonsillar-derived mesenchymal stem cells or a culture thereof for enhancing leukocyte function or a method for screening tonsil-derived mesenchymal stem cells or a culture solution for enhancing immunity.

The amygdala-derived mesenchymal stem cells or their culture medium are characterized in that the expression level of PCOLCE2 is high (eg, above the above-mentioned standard value).

The present invention also provides a pharmaceutical composition comprising tonsillar-derived mesenchymal stem cells or a culture thereof selected according to a method for screening tonsil-derived mesenchymal stem cells or a culture thereof for enhancing leukocyte function or a method for screening tonsil-derived mesenchymal stem cells or a culture thereof for enhancing immunity.

The present invention also provides a composition for preventing or treating leukopenia comprising tonsil-derived mesenchymal stem cells or a culture medium thereof.

The present invention also provides an adjuvant for bone marrow transplantation or anticancer treatment comprising tonsil-derived mesenchymal stem cells or a culture medium thereof, selected according to the above-mentioned screening method.

In the pharmaceutical composition of the present invention, the tonsil-derived mesenchymal stem cells or their culture medium exhibits remarkable effects in the prevention or treatment of leukopenia through their administration. Administration in combination with leukocytes is also possible, if desired. In particular, the tonsil-derived mesenchymal stem cells or their culture medium selected according to the method of the present invention greatly enhances the function of leukocytes (the ROS generating ability of neutrophil cells) and minimizes cell diversity, thereby exhibiting more excellent therapeutic effects without side effects.

The composition of the present invention may be provided in the form of a combination.

The composition may further include a pharmaceutically acceptable carrier.

The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

In addition, if necessary, the composition of the present invention may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, and fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, It may be used by adding additional binders or lubricants.

The composition of the present invention may be formulated and used in various dosage forms suitable for oral or parenteral administration, but more preferably, it may be formulated into a dosage form for parenteral administration, more preferably, it may be formulated into an injection or injection.

In order to formulate the composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, external preparations, etc. may be used, and propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvents or suspensions. In addition, when formulated as an injection or infusion, the composition of the present invention may be mixed in water with a stabilizer or buffer to prepare a solution or suspension, which may be formulated for unit administration in an ampoule or vial.

In addition, it can be transplanted and administered using an administration method commonly used in the art, preferably directly engrafted or transplanted to the diseased area of a patient in need of treatment, but is not limited thereto. For example, the composition of the present invention can be administered rectal, subcutaneous, intramuscular, intraperitoneal, intravenous, arterial, intracerebrospinal, intramedullary, etc., preferably into the bone marrow. In addition, the administration can be carried out by both non-surgical administration using a catheter and surgical administration methods such as injection or transplantation after incision in the diseased area.

The composition of the present invention can be administered at the same time as bone marrow transplantation or chemotherapy or at an appropriate time (including before or after bone marrow transplantation) so that transplanted or to-be-transplanted leukocytes (neutrophil cells) can engraft quickly and smoothly in an individual suffering from leukopenia.

The composition of the present invention may include the tonsil-derived mesenchymal stem cell culture medium at a concentration of 0.4 mg/ml to 1 mg/ml, or the tonsil-derived mesenchymal stem cells at a concentration of 1.0 X 10 ⁵ cells/ml to 1.0 X 10 ⁹ cells/ml, specifically, 1.0 X 10 ⁶ cells/ml to 1.0 X 10 ⁸ cells/ml.

In addition, if necessary, white blood cells, preferably neutrophil cells, may be included in an amount of 1.0 X 10 ⁵ cells/ml to 1.0 X 10 ⁹ cells/ml, specifically, 1.0 X 10 ⁶ cells/ml to 1.0 X 10 ⁸ cells/ml.

When the leukocytes are administered at the same time or at the same time as the tonsil-derived mesenchymal stem cells or the culture medium thereof, the doses of the tonsil-derived mesenchymal stem cells or the culture medium thereof and the leukocytes can be appropriately adjusted to sufficiently promote engraftment of bone marrow cells. It should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and sex, and therefore, the dosage is not limited in any way to the scope of the present invention.

The effective amount and effective dosage of the composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration, etc., and may vary depending on the type and degree of response to be achieved by administration of the composition, the type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, various factors including components of other compositions used simultaneously or at the same time in the subject, and similar factors well known in the pharmaceutical field, conventional knowledge in the art A person who has this can easily determine the dosage so that the desired effect can be sufficiently exerted.

In the present invention, "a subject requiring bone marrow transplantation or chemotherapy" is, but is not limited to, patients suffering from leukopenia diseases such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, aplastic anemia, Fanconi anemia, immunodeficiency, multiple myeloma, myeloproliferative tumor, myelodysplastic syndrome, Hodgkin's lymphoma, etc. may include all individuals who are damaged and require bone marrow cell transplantation.

In the present invention, the bone marrow transplantation or chemotherapy adjuvant refers to an agent that can be used as an adjuvant to enhance the effect of bone marrow transplantation or chemotherapy commonly used in the art. By using the adjuvant according to the present invention, neutrophil cells can promote ROS generation, thereby enhancing the reduced immunity after bone marrow transplantation or chemotherapy in a subject in need of bone marrow transplantation or chemotherapy. The transplant adjuvant of the present invention may be administered simultaneously with or at an appropriate time (including before or after bone marrow transplantation or chemotherapy) with bone marrow transplantation or chemotherapy so that neutrophils can rapidly and smoothly generate ROS in an individual suffering from leukopenia, specifically in need of bone marrow transplantation or chemotherapy.

The preparation for measuring the expression level of mRNA or protein of PCOLCE2 according to the present invention can solve the problem of heterogeneity according to the combination of various precursor cells (precursor cell), so as to solve the problem of tonsil-derived mesenchymal stem cells or their culture. It provides information on the effect of white blood cell function enhancement. Through this, it is possible to determine tonsillar-derived mesenchymal stem cells or their culture medium suitable for administration as a combination or adjuvant to bone marrow transplantation or chemotherapy to enhance immunity, and by using this, it can be used for the development of therapeutic agents that can be applied to patients with a reduced number of white blood cells, such as a state in which immunity is reduced after bone marrow transplantation or chemotherapy.

1 is a result of confirming the ability of each group to generate reactive oxygen species by co-cultivating tonsil-derived mesenchymal stem cells (TMSC) and bone marrow-derived stem cells (BMSC) and neutrophil cells, respectively.
2 is a result of confirming the reactive oxygen species generating ability of tonsillar-derived mesenchymal stem cells according to the difference in donors.
3 is a result of confirming the difference in generation of reactive oxygen species between a group in which generation of reactive oxygen species is increased and a group in which generation is decreased.
4 is a result of measuring the expression levels of SFRP4, MEDAG, MFAP5, and PCOLCE2 genes in the active oxygen species generation increasing group and the decreasing group through real-time PCR.
5 shows the result of confirming PCOLCE2 protein expression in TMSC cells (left side) and the result of confirming PCOLCE2 expression in the actual TMSC culture medium (conditioned medium) (right side).
6 is a result of confirming the expression of PCOLCE2 through real-time PCR after treating tonsil-derived mesenchymal stem cells with excellent reactive oxygen species generating ability with siRNA.
7 is a result of measuring reactive oxygen species by treating tonsil-derived mesenchymal stem cells with siRNA to reduce PCOLCE2 expression and then co-cultivating them with dHL-60 cells.
8 is a result of measuring reactive oxygen species by treating dHL-60 cells with PCOLCE2 at each concentration.
9 shows the expression of NOX3, NOX4, NOX5, and DUOX2 genes involved in leukocyte ROS generation according to PCOLCE2 treatment, expression of PADI4 gene involved in NET formation, and expression of RAC2 gene involved in phagocytosis.

Examples are presented to aid understanding of the present invention. The following examples are only provided to more easily understand the present invention, but the content of the present invention is not limited by the examples.

<Example 1> Establishment of tonsil-derived mesenchymal stem cells

Tonsil-derived mesenchymal stem cells (TMSC) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Specifically, tonsil-derived mesenchymal stem cells were obtained from a total of 25 different donors, and they were separately stored and cultured by dividing them by donor.

On the other hand, human bone marrow-derived stem cells (BMSC) were obtained from PromoCell (Heidelberg, Germany), American Type Culture Collection (Manassas, VA, USA) and Severance Hospital Cell Therapy Center (Seoul, Korea), and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The medium was changed every 2 to 3 days, and cells of passage 5 to 10 were used.

Meanwhile, HL-60 cells, a whole bone marrow cell line, were purchased from American Type Culture Collection and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

In addition, for differentiation of HL-60 cells into granulocyte-like cells, HL-60 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and 0.8% N,N-dimethylformamide (DMF) for 4 days. Then, differentiation was confirmed by flow cytometry, and human neutrophil cells were modeled using differentiated HL-60 (dHL-60) cells.

<Example 2> Co-culture of stem cells and neutrophil cells

In order to investigate the effect of tonsil-derived mesenchymal stem cells (TMSC) and bone marrow-derived stem cells (BMSC) on neutrophil function, TMSC and BMSC prepared in Example 1 were each cultured with neutrophil cells.

Specifically, TMSC and BMSC were seeded at a cell density of 5×10 ⁵ in a 100 mm culture dish, and then dHL-60 cells were added dropwise thereon at a cell density of 1×10 ⁶ and co-cultured for 48 hours. Then, the co-cultured cells were incubated in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C. for 24 hours.

As a control, HL-60 cells and dHL-60 cells, respectively, were added to DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at a density of 1×10 ⁶ cells.

Only floating cells from dHL-60 cells from each experimental group were carefully harvested, and ROS production from dHL-60 cells was stimulated by treatment with 1 nM of phorbol 12-myristate 13-acetate (sigma Aldrich), a pharmacological NADPH oxidase (NOX) activator. Thereafter, Giemsa staining was performed according to the manufacturer's protocol (Abcam, Cambridge, United Kingdom) to visualize the cells under a microscope, and the results are shown in FIG. 1 .

As can be seen in Figure 1, PMA stimulation increased the ROS level in dHL-60 cells, and the ROS in the group co-cultured with TMSC and BMSC and dHL-60 showed a higher level of increase than that in HL-60 and dHL-60 cells. In particular, it was confirmed that the increase in reactive oxygen species was most excellent in the group in which TMSC and dHL-60 were co-cultured.

<Example 3> Evaluation of reactive oxygen species generating ability of tonsil-derived mesenchymal stem cells for each donor

In order to determine whether there is a donor mutation to be overcome in TMSC for the development of a standard cell therapy, the reactive oxygen species generating ability of each donor of tonsillar-derived mesenchymal stem cells, which were confirmed to have a high reactive oxygen species enhancing effect in Example 2, was evaluated.

Specifically, the reactive oxygen species generating ability of tonsillar-derived mesenchymal stem cells from a total of 25 different donors (numbered as TMSC.No) prepared in Example 1 was confirmed in the same manner as in Example 2, and the results are shown in FIGS. 2 and 3.

As can be seen in FIG. 2 , most of the TMSCs improved ROS production in dHL-60 cells stimulated with PMA, but the degree of improvement was different depending on the donor. That is, it was confirmed that classification of mesenchymal stem cells suitable for generating high reactive oxygen species is necessary because there is a diversity of stem cells depending on the donor.

Accordingly, a group with increased production of reactive oxygen species and a reduced production group were selected and characterized among tonsil-derived mesenchymal stem cells. As a result, the group with increased production of reactive oxygen species was TMSC6, 3, 9, 2, 4, and 17, which increased reactive oxygen species by 170% or more, and the group with reduced production of reactive oxygen species was TMSC25, 16, 14, 5, It was 19, 22, 20.

When comparing the increase in generation of reactive oxygen species in each group selected as described above, as can be seen in FIG. 3 , it was confirmed that the generation of reactive oxygen species in the group with increased generation of reactive oxygen species increased nearly twice.

<Example 4> RNAseq and gene expression analysis of tonsil-derived mesenchymal stem cells by donor

In order to confirm the difference in gene expression patterns between the high ROS group and the low ROS group, RNA sequencing (RNAseq) was analyzed for each donor's characteristics of tonsillar-derived mesenchymal stem cells.

As shown in Table 1, four upregulated genes were identified in the high ROS group with thresholds set at “p < 0.05, fold change > 1.5” compared to the low ROS group. On the other hand, as shown in Table 2, the downregulated genes with the threshold set at "p < 0.05, fold change < -2" were identified as 6 in the high ROS group compared to the low ROS group.

Specifically, genes with fold change 2 or more were selected as active oxygen production increasing genes, and as a result, it was confirmed that four genes, sFRP4, MEDAG, MFAP5, and PCOLCE2, were increased in the active oxygen species increased group compared to the active oxygen species lowered group.

dHL-60 Reactive Oxygen Generation Increased Group Gene Symbol Description High/Low ROS generation (Fold change) sFRP4 secreted frizzled related protein 4 2.656180 MEDAG mesenteric estrogen dependent adipogenesis 2.580794 MFAP5 microfibrillar associated protein 5 2.356594 PCOLCE2 procollagen C-endopeptidase enhancer 2 2.270995

dHL-60 reactive oxygen species production reduction group Gene Symbol Description High/Low ROS generation (Fold change) APCDD1 APC down-regulated 1 -2.631378 COL8A1 collagen type VIII alpha 1 chain -2.384816 PSG1 pregnancy specific beta-1 glycoprotein 1 -2.248349 PDPN podoplanin -2.150235 SNAP25 synaptosome associated protein 25 -2.038579 PUNX3 runt related transcription factor 3 -2.022428

On the other hand, the alteration of the above gene was further confirmed using real-time PCR.

Specifically, the expression levels of four genes, SFRP4, MEDAG, MFAP5, and PCOLCE2, were measured in the ROS generation increasing group and the ROS reducing group.

As shown in FIG. 4, the SFRP4 and PCOLCE2 genes with significant results were selected as target proteins contributing to the increase in reactive oxygen species production in TMSC, and among them, PCOLCE2, which had the largest fold change, was finally selected as a candidate gene for enhancing ROS production in dHL-60 cells, and the following experiments were performed.

<Example 5> Confirmation of PCOLCE2 protein expression in cells and culture medium of tonsillar-derived mesenchymal stem cells

PCOLCE2 protein expression was confirmed in cells and culture medium of tonsillar-derived mesenchymal stem cells by real-time PCR.

As shown in FIG. 5, as a result of confirming the expression of PCOLCE2 protein in TMSC cells, the expression of PCOLCE2 protein was increased 1.6±0.1 times in the high ROS group compared to the low ROS group (left), and the expression of PCOLCE2 protein in the TMSC culture medium (conditioned medium) was also confirmed. In the same way, it was confirmed that the secretion of PCOLCE2 protein was increased 1.88±0.28 times in the high ROS group than in the low ROS group.

<Example 6> Confirmation of reactive oxygen species generating ability according to PCOLCE2 expression inhibition

siRNA-mediated gene knockdown was performed to investigate whether elevated PCOLCE2 in the high ROS group plays an important role in TMSCs in enhancing ROS production in dHL-60 cells.

That is, as a result of conducting an experiment to confirm the ability to generate reactive oxygen species according to PCOLCE2 expression inhibition, as shown in FIG. 6 , it was confirmed that transfection of PCOLCE2-targeting siRNA successfully downregulated PCOLCE2 expression.

In addition, as shown in Figure 7, PCOLCE2 gene knockdown prevented the enhancing effect of TMSC on PMA-stimulated ROS production in dHL-60 cells.

<Example 7> Confirmation of reactive oxygen species generating ability according to PCOLCE2 treatment

In order to confirm whether PCOLCE2 is actually involved in increased production of reactive oxygen species, dHL-60 cells were treated with PCOLCE2 recombinant protein at different concentrations, and the increased production of reactive oxygen species was measured.

As shown in FIG. 8, it was confirmed that the production of reactive oxygen species increased as the concentration of PCOLCE2 treatment increased, confirming that PCOLCE2 plays an important role in increasing the production of reactive oxygen species in neutrophils. It was determined that PCOLCE2 would be applicable as a treatment candidate for enhancing immunity as well as reducing morbidity and mortality associated with infection.

<Example 8> Investigation of molecular mechanism according to PCOLCE2 treatment

To elucidate the molecular mechanism for PCOLCE2 enhancement of ROS production in HL-60 cells, genes involved in neutrophil function were investigated using real-time PCR. Specifically, since NOX and double oxidase (DUOX) mainly play essential roles in ROS generation, five isotypes of NOX and two isotypes of DUOX were investigated, and genes involved in the formation of neutrophil extracellular traps (NETs) and phagocytosis were additionally investigated.

As shown in FIG. 9, it was confirmed that the treatment of PCOLCE2 increased the expression of genes NOX3, NOX4, NOX5, DUOX2, genes involved in leukocyte ROS production, PADI4, a gene involved in NET formation, and RAC2, a gene involved in phagocytosis.

In the present specification, details that can be sufficiently recognized and inferred by those skilled in the art of the present invention have been omitted, and in addition to the specific examples described herein, the technical spirit or essential configuration of the present invention is not changed. Various modifications are possible. Accordingly, the present invention may be practiced in a manner different from that specifically described and exemplified herein, which can be understood by those skilled in the art.

Claims (17)

A composition for predicting enhancement of leukocyte function of tonsillar-derived mesenchymal stem cells or their culture medium containing an agent for measuring the expression level of PCOLCE2 mRNA or protein. According to claim 1, wherein the tonsil-derived mesenchymal stem cells are human-derived, tonsillar-derived mesenchymal stem cells or a composition for predicting enhancement of leukocyte function in a culture medium thereof. The composition for predicting enhancement of leukocyte function of tonsillar-derived mesenchymal stem cells or their culture medium according to claim 1, wherein the agent for measuring the mRNA level is a primer or a probe. The composition for predicting enhancement of leukocyte function of tonsillar-derived mesenchymal stem cells or their culture medium according to claim 1, wherein the agent for measuring the protein level is a polyclonal antibody, a monoclonal antibody or a recombinant antibody that specifically binds to PCOLCE2. According to claim 1, wherein the leukocyte function is ROS production ability of neutrophil cells, tonsil-derived mesenchymal stem cells or a composition for predicting enhancement of leukocyte function in its culture medium. A composition for screening tonsillar-derived mesenchymal stem cells or their culture medium for enhancing immunity, comprising an agent for measuring the expression level of PCOLCE2 mRNA or protein. The method of claim 6, wherein the tonsil-derived mesenchymal stem cells are human-derived, tonsillar-derived mesenchymal stem cells for immune enhancement or a composition for screening a culture medium thereof. [Claim 7] The composition for screening tonsillar-derived mesenchymal stem cells or their culture medium for enhancing immunity according to claim 6, wherein the agent for measuring the mRNA level is a primer or a probe. The composition for screening tonsil-derived mesenchymal stem cells or their culture medium for enhancing immunity according to claim 6, wherein the agent for measuring the protein level is a polyclonal antibody, a monoclonal antibody or a recombinant antibody that specifically binds to PCOLCE2. The method of claim 6, wherein the immune enhancement is caused by enhancing the ROS-producing ability of neutrophil cells, tonsillar-derived mesenchymal stem cells or a composition for screening a culture medium thereof for enhancing immunity. A kit comprising a composition for predicting enhancement of leukocyte function of the tonsil-derived mesenchymal stem cells or their culture medium according to any one of claims 1 to 5. A kit comprising the composition for screening tonsillar-derived mesenchymal stem cells or their culture medium for enhancing immunity according to any one of claims 6 to 10. (a) obtaining a sample from tonsillar-derived mesenchymal stem cells or their culture; (b) measuring the expression level of PCOLCE2 mRNA or protein in the sample; And (c) selecting tonsillar-derived mesenchymal stem cells or their culture medium in which the expression level of PCOLCE2 in step (b) is increased compared to the reference value. (a) obtaining a sample from tonsillar-derived mesenchymal stem cells or their culture; (b) measuring the expression level of PCOLCE2 mRNA or protein in the sample; And (c) selecting tonsillar-derived mesenchymal stem cells or their culture medium, in which the expression level of PCOLCE2 in step (b) increases compared to the reference value, as tonsil-derived mesenchymal stem cells or their culture medium for immunity enhancement; A method for screening tonsillar-derived mesenchymal stem cells or its culture medium for immunity enhancement using an agent for measuring the expression level of mRNA or protein of PCOLCE2. A pharmaceutical composition for preventing or treating leukopenia, comprising tonsil-derived mesenchymal stem cells selected by the screening method according to claim 13 or 14 or a culture medium thereof. The pharmaceutical composition for preventing or treating leukopenia according to claim 15, wherein the white blood cells are neutrophil cells. The pharmaceutical composition for preventing or treating leukopenia according to claim 15, wherein leukopenia is caused by bone marrow transplantation or chemotherapy.