Screening method for nerve regeneration-promoting cells having nerve regeneration activity

Abstract

The present invention relates to a method for screening nerve regeneration-promoting cells having nerve regeneration activity derived from mesenchymal stem cells and a pharmaceutical composition containing the nerve regeneration-promoting cells. The nerve regeneration-promoting cells of the present invention have a completely different expression pattern of CD markers compared to stem cells, and have excellent nerve regeneration effects, and can be used in various fields in the prevention or treatment of neurological diseases.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to a method for screening for nerve regeneration-promoting cells having nerve regeneration activity derived from stem cells, and a pharmaceutical composition for preventing or treating neurological diseases, which contains the nerve regeneration-promoting cells.

[0002] [Background technology] Mesenchymal stem cells (MSCs) have the differentiation flexibility to differentiate into other cells in response to specific stimuli. They are free from the potential for tumorigenesis associated with dedifferentiated stem cells and the ethical issues associated with embryonic stem cells, making them widely used in the development of cell therapy drugs. Mesenchymal stem cells are adult stem cells that are typically isolated from tissues such as adult fat, umbilical cord blood, and bone marrow. Isolation methods for these tissues are invasive, painful, and have limitations in that they do not yield large numbers of stem cells.

[0003] Summary of the Invention [Problem to be solved by the invention] Therefore, the inventors have developed a method for screening nerve regeneration-promoting cells that exhibit nerve regeneration effects by analyzing specific CD markers of cells that have differentiated into nerve regeneration-promoting cells from various cells differentiated from mesenchymal stem cells, and have completed the present invention.

[0004] Therefore, an object of the present invention is to provide a method for screening for neuronal regeneration promoting cells having neuronal regeneration activity derived from mesenchymal stem cells.

[0005] Another object of the present invention is to provide nerve regeneration-promoting cells screened by the screening method.

[0006] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurological disease, which comprises the nerve regeneration-promoting cells as an active ingredient.

[0007] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings.

[0008] [Means for solving the problem] According to one aspect of the present invention, there is provided a method for screening for neuronal regeneration promoting cells having neuronal regeneration activity derived from mesenchymal stem cells, comprising the following steps: i) providing cells differentiated from mesenchymal stem cells; and ii) A step of selecting cells from the differentiated cells of step i) in which one or more markers selected from the group consisting of CD121a, CD106 and CD112 are up-regulated compared to pre-differentiated mesenchymal stem cells.

[0009] According to another aspect of the present invention, there is provided a method for screening for neuronal regeneration promoting cells having neuronal regeneration activity derived from mesenchymal stem cells, comprising the steps of: i) providing cells differentiated from mesenchymal stem cells; and ii) A step of selecting cells from the differentiated cells of step i) in which one or more markers selected from the group consisting of CD26 and CD141 are down-regulated compared to mesenchymal stem cells before differentiation.

[0010] The inventors differentiated mesenchymal stem cells of various origins and examined the expression patterns of numerous markers in these differentiated cells. As a result, they surprisingly found that cells differentiated into nerve regeneration-promoting cells showed common tendencies in the expression patterns of certain markers (e.g., CD markers such as CD121a, CD106, and CD112).

[0011] As used herein, the term "neuronal regeneration promoting cell" or "NRPC" refers to a cell differentiated from a mesenchymal stem cell and possessing a neuroregenerative effect (e.g., the effect of directly or indirectly promoting neuroregeneration in structural or functional terms by myelination of peripheral nerves in damaged neurons or secreting cytokines necessary for neuroregeneration).

[0012] According to a preferred embodiment of the present invention, the screening method may include the following steps: i) preparing differentiated cells from mesenchymal stem cells; ii) selecting cells from the differentiated cells of step i) in which one or more markers selected from the group consisting of CD121a, CD106, and CD112 are up-regulated compared to mesenchymal stem cells before differentiation; and iii) A step of selecting cells from the differentiated cells of step i) in which one or more markers selected from the group consisting of CD26 and CD141 are down-regulated compared to mesenchymal stem cells before differentiation.

[0013] According to a preferred embodiment of the present invention, the screening method may include the following steps: i) preparing differentiated cells from mesenchymal stem cells; ii) selecting cells from the differentiated cells of step i) in which one or more markers selected from the group consisting of CD26 and CD141 are down-regulated compared to mesenchymal stem cells before differentiation; and iii) A step of selecting cells from the differentiated cells of step i) in which one or more markers selected from the group consisting of CD121a, CD106 and CD112 are up-regulated compared to pre-differentiated mesenchymal stem cells.

[0014] In the present invention, the term "stem cell" refers to a cell that has the ability to self-renew and differentiate into two or more cells, and the stem cell includes adult stem cells, pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells, and is preferably a mesenchymal stem cell.

[0015] In the present invention, the term "mesenchymal stem cells" refers to undifferentiated stem cells isolated from human or mammalian tissue. Mesenchymal stem cells can be derived from various tissues, particularly from one or more tissues selected from the group consisting of tonsil, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorion, decidua, and placenta. Techniques for isolating stem cells from each tissue are already known in the art.

[0016] According to a preferred embodiment, the mesenchymal stem cells are derived from tonsils or adipose tissue.

[0017] According to one embodiment of the present invention, it has been found that it is most preferable to use mesenchymal stem cells derived from tonsils or fat.

[0018] As used herein, the term "CD" or "cluster of differentiation" molecule refers to a surface molecular structure present on the cell surface. Some CD molecules are commonly expressed in different cell populations and are used to distinguish cell populations (i.e., as markers). Cells of the same lineage have the same type of CD molecule, and even within the same cell population, different CD molecules are present depending on the differentiation or activation stage. This is useful for identifying the lineage, differentiation stage, and activation of cells.

[0019] According to one embodiment of the present invention, by comparing the CD molecule expression patterns of nerve regeneration-promoting cells having nerve regeneration activity derived from stem cells of the present invention with those of mesenchymal stem cells, it was demonstrated that the nerve regeneration-promoting cells of the present invention are different from mesenchymal stem cells. Furthermore, in the present invention, CD10, CD39, CD106, CD112, CD121a, CD338, etc., whose expression is upregulated compared to mesenchymal stem cells, and CD26, CD54, CD126, CD141, etc., whose expression is downregulated, can be used as differentiation markers for nerve regeneration-promoting cells.

[0020] According to a preferred embodiment of the present invention, the differentiated cells in step i) are obtained by culturing mesenchymal stem cells to form neurospheres, which are then differentiated.

[0021] According to a preferred embodiment, the nerve regeneration activity includes myelination of peripheral nerves.

[0022] In the present invention, the term "myelination" refers to the phenomenon in which myelin sheaths wrap around axons of peripheral nerves, accelerating the transmission of stimuli. Injured peripheral nerves are normalized (i.e., regenerated) through myelination.

[0023] According to one embodiment of the present invention, some of the candidate cells screened by the screening method of the present invention were morphologically myelinated through a dorsal root ganglion co-culture process.

[0024] In yet another aspect, the present invention provides nerve regeneration-promoting cells screened by the above screening method.

[0025] According to a preferred embodiment of the present invention, the nerve regeneration-promoting cells have the following characteristics: a) expression of the markers CD121a, CD106, and CD112 is upregulated compared to predifferentiated mesenchymal stem cells; and b) Expression of the markers CD26 and CD141 is downregulated compared to pre-differentiated mesenchymal stem cells.

[0026] In the nerve regeneration-promoting cells, the expression of the marker CD121a is upregulated by preferably 30% or more, more preferably 40% or more, compared to that of mesenchymal stem cells before differentiation.

[0027] According to one embodiment of the present invention, compared to pre-differentiated mesenchymal stem cells, the expression of the marker CD121a was upregulated by 94% in T-MSC-1-1-derived nerve regeneration-promoting cells, 71% in T-MSC-1-2-derived nerve regeneration-promoting cells, 51% in T-MSC-1-3-derived nerve regeneration-promoting cells, and 48% in T-MSC-1-4-derived nerve regeneration-promoting cells, an average of more than 66%.

[0028] In the nerve regeneration-promoting cells, the expression of the marker CD106 is upregulated by preferably 5% or more, more preferably 10% or more, compared to that of mesenchymal stem cells before differentiation.

[0029] According to one embodiment of the present invention, compared to pre-differentiated mesenchymal stem cells, the expression of the marker CD106 was upregulated by 30% in T-MSC-1-1-derived nerve regeneration-promoting cells, 11% in T-MSC-1-2-derived nerve regeneration-promoting cells, 16% in T-MSC-1-3-derived nerve regeneration-promoting cells, and 13% in T-MSC-1-4-derived nerve regeneration-promoting cells, an average of more than 17%.

[0030] In the nerve regeneration-promoting cells, the expression of the marker CD112 is upregulated preferably by 10% or more, more preferably by 15% or more, compared to that of mesenchymal stem cells before differentiation.

[0031] According to one embodiment of the present invention, compared to pre-differentiated mesenchymal stem cells, the expression of the marker CD112 was upregulated by 49% in T-MSC-1-1-derived nerve regeneration-promoting cells, 25% in T-MSC-1-2-derived nerve regeneration-promoting cells, 30% in T-MSC-1-3-derived nerve regeneration-promoting cells, and 19% in T-MSC-1-4-derived nerve regeneration-promoting cells, an average of more than 30%.

[0032] According to a preferred embodiment of the present invention, the nerve regeneration-promoting cells have down-regulated expression of the marker CD26 compared to pre-differentiation mesenchymal stem cells.

[0033] In the nerve regeneration-promoting cells, the expression of the marker CD26 is downregulated preferably by 5% or more, more preferably by 8% or more, compared to that of mesenchymal stem cells before differentiation.

[0034] According to one embodiment of the present invention, compared to pre-differentiated mesenchymal stem cells, the expression of the marker CD26 was down-regulated by 9% in T-MSC-1-1-derived nerve regeneration-promoting cells, 11% in T-MSC-1-2-derived nerve regeneration-promoting cells, 27% in T-MSC-1-3-derived nerve regeneration-promoting cells, and 16% in T-MSC-1-4-derived nerve regeneration-promoting cells, an average of more than 16%.

[0035] In the nerve regeneration-promoting cells, the expression of the marker CD141 is downregulated preferably by 5% or more, more preferably by 8% or more, compared to that of mesenchymal stem cells before differentiation.

[0036] According to one embodiment of the present invention, compared to pre-differentiation mesenchymal stem cells, the expression of the marker CD141 was down-regulated by 9% in T-MSC-1-1-derived nerve regeneration-promoting cells, 20% in T-MSC-1-2-derived nerve regeneration-promoting cells, 16% in T-MSC-1-3-derived nerve regeneration-promoting cells, and 38% in T-MSC-1-4-derived nerve regeneration-promoting cells, an average of more than 20% down-regulation.

[0037] In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating a neurological disease, which comprises the nerve regeneration-promoting cells as an active ingredient.

[0038] According to yet another aspect of the present invention, there is provided a method for treating a neurological disease, comprising the step of administering an effective amount of the nerve regeneration-promoting cells to a subject.

[0039] According to yet another aspect of the present invention, the present invention provides a therapeutic use of the nerve regeneration-promoting cells.

[0040] In the present invention, the term "neurological disease" refers to a disease caused by damage to nervous tissue due to internal factors such as heredity or aging, or external factors such as trauma.

[0041] According to a preferred embodiment of the present invention, the neurological disease is one or more diseases selected from the group consisting of Charcot-Marie-Tooth neuropathy, diabetic peripheral neuropathy, spinal cord injury, amyotrophic lateral sclerosis, carpal tunnel syndrome, polio, leprosy, muscular dystrophy, polymyositis, and myasthenia gravis.

[0042] In the present invention, the term "subject" refers to an individual to which the composition of the present invention or the nerve regeneration-promoting cells needs to be administered, and is not limited to administration subjects such as mammals, birds, reptiles, amphibians, and fish.

[0043] In the present invention, "prevention" refers to any action that suppresses or delays a neurological disorder by administering the composition of the present invention, and "treatment" refers to any action that improves or favorably alters the symptoms of a neurological disorder by administering the composition of the present invention.

[0044] According to a preferred embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier or excipient.

[0045] The pharmaceutical composition of the present invention may be prepared in unit dose form or in a multi-dose container by formulating it with pharmaceutically acceptable carriers and / or excipients by a method that can be easily carried out by a person skilled in the art to which the invention pertains.

[0046] The pharmaceutical composition according to the present invention can be formulated into various forms by conventional methods and used, for example, into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and into external preparations, suppositories, and sterile injection solutions.

[0047] The composition of the present invention may contain one or more known active ingredients that have preventive or therapeutic effects on neurological diseases, in addition to nerve regeneration-promoting cells that are derived from stem cells and have nerve regeneration activity.

[0048] The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, and can be administered by, for example, intravenous injection, transdermal injection, subcutaneous injection, intramuscular injection, intravitreal injection, subretinal injection, suprachoroidal injection, eye drop administration, intraventricular injection, intrathecal injection, intraamniotic injection, intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisternal injection, intracoronary injection, intracranial injection, intradural injection, or epidural injection. injection), intrahippocampal injection, intranasal injection, intraosseous injection, intraperitoneal injection, intrapleural injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injectionIt can be administered by intratumoral injection, local injection, intraperitoneal injection, and the like.

[0049] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0050] The dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, mode of administration, administration time and / or route of administration of the pharmaceutical composition, and on various factors including the type and degree of response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms and degree of disease of the individual to be administered, sex, diet, excretion, drugs administered to the individual simultaneously or at different times, other components of the composition, etc., as well as similar factors well known in the medical field. A person of ordinary skill in the art can easily determine and prescribe an effective dosage for the intended treatment.

[0051] The administration route and mode of the pharmaceutical composition of the present invention may be independent of each other and are not particularly limited. Any administration route and mode may be used as long as the pharmaceutical composition can reach the desired site.

[0052] [Effects of the Invention] The features and advantages of the present invention can be summarized as follows: (i) The present invention provides a method for screening for nerve regeneration-promoting cells having nerve regeneration activity derived from mesenchymal stem cells, and a pharmaceutical composition containing the nerve regeneration-promoting cells.

[0053] (ii) The nerve regeneration-promoting cells of the present invention have a completely different CD marker expression pattern compared to mesenchymal stem cells and possess nerve regeneration effects, making them suitable for a variety of uses in the prevention and treatment of neurological diseases.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Photographs showing the results of inducing T-MSC-1-1, a tonsil-derived mesenchymal stem cell, into nerve regeneration-promoting cells on a daily basis.

[0055] [Fig. 2] A heat map showing the CD marker expression of the nerve regeneration-promoting cells according to the present invention, as determined by CD marker screening.

[0056] [Figure 3] Screening results for CD markers with different expression levels in nerve regeneration-promoting cells compared to tonsil-derived mesenchymal stem cells. Figure 3A shows the results of a comparison of CD markers with increased expression levels compared to tonsil-derived mesenchymal stem cells, and Figure 3B shows the results of a comparison of CD markers with decreased expression levels compared to tonsil-derived mesenchymal stem cells.

[0057] [Figure 4] Comparison of the expression patterns of CD markers whose expression was increased and decreased in nerve regeneration-promoting cells compared to tonsil-derived mesenchymal stem cells.

[0058] [FIG. 5] Histogram showing the results of screening for CD markers whose expression was commonly increased or decreased in nerve regeneration-promoting cells.

[0059] [FIG. 6] The expression patterns of markers CD121a, CD106, and CD112, which were commonly increased in nerve regeneration-promoting cells, are compared with those of tonsil-derived mesenchymal stem cells.

[0060] [Fig. 7] The expression patterns of markers CD26 and CD141, which are commonly decreased in nerve regeneration-promoting cells, are compared with those of tonsil-derived mesenchymal stem cells.

[0061] [Figure 8] This figure shows the results of comparing the expression of CD markers in tonsil-derived mesenchymal stem cells (T-MSCs) and nerve regeneration-promoting cells (NRPCs) in a heat map to compare their CD marker expression patterns.

[0062] [Figure 9] Results of a neurite outgrowth assay performed to measure and compare the growth of neurites from nerve regeneration-promoting cells.

[0063] [Figure 10] These are the results confirming that the process of co-culturing candidate cells with dorsal root ganglia results in myelination in some cells morphologically.

[0064] [Figure 11] Flow cytometry analysis of up- and down-regulated CD markers selected in the CD screening using individual antibodies.

[0065] Figure 12 shows the results of cytokine array analysis of T-MSCs and NRPCs as a heat map (left), and the results of dividing the cytokines that were commonly increased in NRPCs compared to T-MSCs by ratio (right) (fold change: NRPC1-1 / T-MSC1-1, NRPC1-2 / T-MSC1-2).

[0066] [Mode for Carrying Out the Invention] The present invention will be described in more detail below using examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0067] [Example] Example 1. Preparation of mesenchymal stem cells 1-1. Isolation and culture of tonsil-derived mesenchymal stem cells Tonsil tissue from multiple donors provided by Ewha Women's University College of Medicine was divided into left and right sections. The tonsil tissue was placed in a tube containing 10 ml of Dulbecco's Phosphate-Buffered Saline (DPBS) supplemented with 20 μg / ml of gentamicin and centrifuged at 1,500 rpm for 5 minutes before being washed. This tissue washing process was repeated twice. The washed tonsil tissue was then cut into small pieces using sterilized scissors.

[0068] To isolate tonsil-derived mesenchymal stem cells from tonsil tissue, an equal weight of enzyme reaction solution was added to the pulverized tonsil tissue and then cultured in a shaking incubator at 37°C and 200 rpm for 60 minutes. The composition of the enzyme reaction solution is shown in Table 1.

[0069] [Table 1]

[0070] The culture medium was mixed with 5% FBS (fetal bovine serum) and centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the pellet was resuspended in 30 ml of DPBS and centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the pellet was resuspended in 10 ml of DPBS to prepare a suspension. The suspension was passed through a 100 μm filter. The tonsil-derived mesenchymal stem cells remaining in the filter were washed with 20 ml of DPBS and centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and ACK lysis buffer was added and incubated in a 37°C water bath for 5 minutes. DPBS was added to the suspension, and the suspension was centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the pellet was resuspended in high-glucose DMEM medium (10% FBS, 20 μg / ml gentamicin) to prepare a cell suspension. The cells in the prepared cell suspension were counted. The cell suspension was seeded into a T175 flask and cultured in an incubator at 37°C and a carbon dioxide concentration of 5%.

[0071] 1-2. Isolation and culture of adipose-derived mesenchymal stem cells Adipose-derived mesenchymal stem cells were purchased from LONZA (Human Adipose-Derived Stem Cells, Cat#PT-5006, Lonza, Switzerland). The purchased adipose-derived mesenchymal stem cells were cultured using a culture medium provided by LONZA (Bulletkit ADSD, Cat#PT-4505).

[0072] Example 2. Neurosphere formation The mesenchymal stem cells described in Example 1 were cultured to form neurospheres. Specifically, mesenchymal stem cells were subcultured to prepare mesenchymal stem cells at passages 4 to 7. After removing the culture medium, the mesenchymal stem cells were washed with DPBS. The washed cells were treated with TrypLE to harvest the cells, and the harvested cells were counted. The harvested cells were centrifuged to remove the supernatant, and then resuspended in a neurosphere-forming medium. The composition of the neurosphere-forming medium is shown in Table 2.

[0073] [Table 2]

[0074] The cells resuspended in neurosphere formation medium were seeded onto a 60 mm ultra-low attachment dish at 1 x 106 cells. The seeded cells were cultured for 3 days at 37°C in 5% carbon dioxide. After 3 days of culture, the neurospheres formed in the dish were collected into a 15 ml tube. The collected cells were centrifuged, the supernatant was removed, and fresh neurosphere formation medium was added to resuspend the cells, producing a neurosphere suspension. The neurosphere suspension was transferred to a low attachment dish, and the neurospheres were cultured for 4 days at 37°C in 5% carbon dioxide.

[0075] Example 3. Differentiation of neurospheres into candidate cells for neuronal regeneration promoting cells (NRPCs) The neurospheres generated in Example 2 were homogenized using a 23-26G syringe needle. The pulverized neurospheres were transferred to a 15 ml tube using a pipette, centrifuged, and the supernatant was removed. Nerve regeneration-promoting cell induction medium was added to the tube, and the pulverized neurospheres were resuspended. The nerve regeneration-promoting cell induction medium is composed of GlutaMAX-containing DMEM / F12 supplemented with: 1) 5-20% FBS (Fetal Bovine Serum), 2) 5-20 ng / ml bFGF (Peprotech, USA), 3) 100-400 μM butylated hydroxyanisole (Sigma, USA), 4) 5-40 μM forskolin (MedCheExpress, USA), 5) 0.1-10% N2 supplements (Gibco, USA), 6) 1-100 ng / ml brain-derived neurotrophic factor (BDNF, Sigma-Aldrich, USA), 7) 1-100 ng / ml nerve growth factor (NGF, Santa Cruz, USA), and 8) 0.01-1 ng / ml sonic hedgehog (Sonic Hedgehog). Various media were prepared by combining three or more of the following: 1) 1) hedgehog (SHH, R&D Systems, USA); 2) 1-10 ng / ml PDGF-AA (Platelet Derived Growth Factor-AA, Peprotech, USA); and 3) 50-300 ng / ml Heregulin-beta 1 (Peprotech, USA).

[0076] The neurospheres resuspended in the various media were seeded onto laminin (2 μg / ml)-coated T175 flasks. The seeded neurospheres were cultured for 8–10 days, and the respective neural regeneration-promoting cell-induction media was replaced every 3 days (Figure 1).

[0077] Example 4. Primary screening of candidate nerve regeneration-promoting cells using confirmation of peripheral nerve myelination The function of peripheral nerve myelination was examined for the candidate nerve regeneration-promoting cells prepared in Example 3. Specifically, to examine myelination in the co-culture of differentiated candidate nerve regeneration-promoting cells and dorsal root ganglia (DRG), candidate cells were co-cultured with DRG.

[0078] Dorsal root ganglion (DRG) cells isolated from rats were purchased from LONZA (Rat Dorsal Root Ganglion Cells, Cat# R-DRG-505, Lonza, Switzerland). The purchased DRG cells were placed on candidate cells and co-cultured. For co-culture, the DRG cells were cultured in the culture medium provided by LONZA (Primary Neuron Growth Medium Bullet Kit (PNGM), Cat# CC-4461).

[0079] The culture medium was changed every 3 days. Morphologically, it was confirmed that some cells were myelinated during the co-culture of the candidate cells and dorsal root ganglia (Figure 10).

[0080] Example 5. Secondary screening using CD marker expression analysis of nerve regeneration-promoting cells Among the nerve regeneration-promoting cell candidate group confirmed to have myelination in Example 4, the expression of a total of 242 CD markers was analyzed for T-MSC-1-1 (tonsil-derived mesenchymal stem cell 1), T-MSC-1-2 (tonsil-derived mesenchymal stem cell 2), T-MSC-1-3 (tonsil-derived mesenchymal stem cell 3), and T-MSC-1-4 (tonsil-derived mesenchymal stem cell 4), which showed the best myelination based on cell morphology, and for the nerve regeneration-promoting cells differentiated from them.

[0081] For CD marker analysis, 3 x 107 target cells were collected. The target cells were washed with DPBS and centrifuged at 2000 rpm for 5 minutes. The supernatant was removed and washed once with DPBS. After centrifugation, the pellet was resuspended in 30 ml of FACS buffer. A round-bottom 96-well plate was prepared and 100 µl (1 x 105 cells) of the cell suspension was dispensed into each well. 10 µl of primary antibody for CD markers was added to each well of the 96-well plate. The plate was incubated on ice for 30 minutes in the dark. 100 µl of FACS buffer was dispensed per well to wash the 96-well plate, followed by centrifugation at 300 g for 5 minutes. After removing the supernatant, 200 µl of FACS buffer was added to each well and centrifuged at 300 g for 5 minutes. A secondary antibody was prepared in FACS buffer at a 1:200 ratio (1.25 µg / ml). After centrifugation, the supernatant was removed, and 100 μl of the prepared secondary antibody was added to each well. The incubation was allowed to continue on ice for 20-30 minutes in the dark. 100 μl of FACS buffer was added to each well to wash, followed by centrifugation at 300 g for 5 minutes. After removing the supernatant, 200 μl of FACS buffer was added to each well to wash the target cells. This washing process was repeated twice. After washing, 200 μl of FACS buffer was added to each well to resuspend the cells, and the expression of CD markers in the target cells was confirmed by flow cytometry (FACS; fluorescence-activated cell sorting).

[0082] The expression of CD markers in the induced nerve regeneration-promoting cells was compared using a heat map, and the results are shown in Figure 2. As shown in Figure 2, the expression patterns of CD markers in nerve regeneration-promoting cells (NRPCs) and mesenchymal stem cells (MSCs) were similar, but the expression patterns of some markers were different.

[0083] We compared the expression patterns of CD markers between mesenchymal stem cells (MSCs) derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 and neural regeneration-promoting cells (NRPCs). CD markers whose expression was increased or decreased are used as differentiation markers for neural regeneration-promoting cells. The CD markers whose expression was increased or decreased are shown in Figure 3.

[0084] As shown in Figure 3A, CD markers whose expression was increased in neural regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 compared to tonsil-derived mesenchymal stem cells included CD10, CD39, CD106, CD112, CD121a, and CD338 (markers that were increased in at least three of the four NRPCs). As shown in Figure 3B, CD markers whose expression was decreased included CD26, CD54, CD126, and CD141 (markers that were decreased in at least three of the four NRPCs).

[0085] The results of comparing the expression increase and decrease rates of CD markers in the tonsil-derived nerve regeneration-promoting cells are shown in FIG.

[0086] As shown in Figure 4, the nerve regeneration-promoting cells derived from T-MSC-1-1 showed increased expression of 12 CD markers and decreased expression of 9 CD markers. The nerve regeneration-promoting cells derived from T-MSC-1-2 showed increased expression of 8 CD markers and decreased expression of 9 CD markers. The nerve regeneration-promoting cells derived from T-MSC-1-3 showed increased expression of 40 CD markers and decreased expression of 3 CD markers. The nerve regeneration-promoting cells derived from T-MSC-1-4 showed increased expression of 17 CD markers and decreased expression of 6 CD markers.

[0087] Based on the results, CD markers whose expression was increased or decreased in all of the nerve regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 were selected. The selected markers are as follows: - Commonly increased CD markers: CD106, CD112, CD121a - Commonly down-regulated CD markers: CD26, CD141 The patterns of the CD markers whose expression was commonly increased and the CD markers whose expression was commonly decreased were also observed in the nerve regeneration-promoting cells differentiated from the adipose-derived mesenchymal stem cells of Example 1-2.

[0088] The results of CD screening of CD markers whose expression was increased or decreased in all of the nerve regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 are shown in a histogram in Figure 5.

[0089] As shown in Figure 5, CD markers CD121a, CD106, and CD112, which commonly show increased expression in nerve regeneration-promoting cells, showed an increase in expression rate of more than 10% after differentiation. On the other hand, markers CD26 and CD141, which commonly show decreased expression, showed a decrease in expression rate of more than about 9% after differentiation. These results suggest that the commonly expressed markers CD121a, CD106, CD112, CD26, and CD141 may be used as differentiation markers for nerve regeneration-promoting cells, and in particular, CD121a, CD106, and CD112 may be used as representative differentiation markers.

[0090] 6-1. Comparison of expression of co-expressed markers CD121a, CD106, and CD112 The expression of the markers CD121a, CD106, and CD112 is increased by more than 10% in nerve regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 compared to mesenchymal stem cells, and is one of the key characteristics of nerve regeneration-promoting cells. Therefore, we compared the expression of the markers CD121a, CD106, and CD112 in nerve regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4, and the results are shown in Figure 6.

[0091] As shown in Figure 6, we confirmed that the expression of the markers CD121a, CD106, and CD112 was significantly increased in neural regeneration-promoting cells (NRPCs) compared to tonsil-derived mesenchymal stem cells (T-MSCs).

[0092] 6-2. Comparison of expression of co-expressed markers CD26 and CD141 The markers CD26 and CD141 were CD markers whose expression was commonly decreased in nerve regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4. The expression of these CD markers was compared in nerve regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4, and the results are shown in Figure 7.

[0093] As shown in FIG. 7, it was confirmed that the nerve regeneration-promoting cells had reduced expression of CD26 and CD141 compared to tonsil-derived mesenchymal stem cells.

[0094] 6-3. Comparison of CD marker expression patterns In order to compare the CD marker expression patterns of tonsil-derived mesenchymal stem cells and nerve regeneration-promoting cells, a heat map was created based on the results of comparing the expression of co-expressed CD markers in Examples 6-1 and 6-2, and is shown in Figure 8.

[0095] As shown in FIG. 8, it was confirmed that the nerve regeneration-promoting cells had different expression of co-expressed markers compared to tonsil-derived mesenchymal stem cells.

[0096] 6-4. Average expression of co-expressed CD markers To compare whether the expression patterns of CD markers in tonsil-derived mesenchymal stem cells and nerve regeneration-promoting cells remain the same after freezing, the expression of co-expressed CD markers was confirmed in Examples 6-1 and 6-2 in live cells before freezing, cells thawed after freezing, and cells cultured with the thawed cells attached, and the average expression rates of co-expressed CD markers are shown in Figure 11.

[0097] As shown in Figure 11, the nerve regeneration-promoting cells showed increased expression of CD106, CD121a, and CD112, and decreased expression of CD26 and CD141, compared to tonsil-derived mesenchymal stem cells, even after freezing. These results confirmed that the nerve regeneration-promoting cells had different co-expression markers compared to tonsil-derived mesenchymal stem cells, regardless of the freezing condition.

[0098] Example 7. Neurite Outgrowth Effect of Nerve Regeneration-Promoting Cells of the Present Invention A neurite (or neuronal process), which is a long extension of one of the dendrites of a nerve cell body, is known to be involved in the transport of substances necessary for axon growth and regeneration, such as neurotransmitters and nerve growth factors (L McKerracher et al., Spinal Cord Repair: Strategies to Promote Axon Regeneration, Neurobiol Dis, 2001). A neurite outgrowth assay was performed to measure and compare the neurite growth of the nerve regeneration-promoting cells of the present invention.

[0099] The above method involves culturing N1E-115 (mouse neuroblastoma cell line, ATCC, USA) and seeding them onto a fine porous filter (Neurite Outgrowth Assay Kit, Millipore, USA). The seeded cells were then cultured for 48 hours in culture medium collected from nerve regeneration-promoting cells and stem cells. The neurites that permeated the fine porous filter and extended were stained and measured for absorbance. The neurite outgrowth assay was used to detect neurites, confirming that the culture medium from the nerve regeneration-promoting cells of the present invention regulated or stimulated neurite (axon) growth in N1E-115 (mouse neuroblastoma), thereby confirming the nerve regeneration effect.

[0100] Therefore, we compared the neurites of nerve regeneration-promoting cells derived from T-MSC-1-2 and N1E-115 cells grown from tonsil-derived stem cell culture medium, and the results are shown in Figure 9 (NRPCs: nerve regeneration-promoting cells derived from T-MSC-1-2, T-MSCs: T-MSC-1-2, Negative control: negative control group, Positive control: positive control group). As a result of the comparison, a larger number of neurites were observed in the nerve regeneration-promoting cells compared to the tonsil-derived stem cells, and absorbance measurements also confirmed that the nerve regeneration-promoting cells had a greater increase in absorbance than tonsil-derived stem cells. (- Negative control: A porous filter (membrane insert provided in the Neurite outgrowth assay kit) was coated with BSA. N1E-115 cells were cultured in culture medium (DMEM medium + 20 μg / ml gentamicin). - Positive control: A porous filter was coated with laminin. N1E-115 cells were cultured in culture medium (DMEM medium + 20 μg / ml gentamicin + 1 mg / ml BSA). - NRPC, T-MSC test group: A porous filter was coated with BSA. N1E-115 cells were cultured in the respective NRPC and T-MSC culture medium.)

[0101] Example 8. Cytokine array analysis of nerve regeneration-promoting cells The expression of 507 cytokines was analyzed for T-MSC-1-1 and T-MSC-1-2, which were the most myelinate-competent cells in Example 4, and the nerve regeneration-promoting cells differentiated from them.

[0102] Target cells were cultured for cytokine analysis. Target cells were seeded in flasks at the same cell number and cultured for 3-4 days. When the flask area covered by target cells reached 80% or more, the existing culture medium was removed and the target cells were washed twice with DPBS. After washing, the medium was replaced with Dulbecco's Phosphate-Buffered Saline (DMEM) without FBS (Fetal Bovine Serum) or cytokines to eliminate the effects of cytokines contained in the culture medium. After culturing the target cells for 30 hours, the culture medium was collected.

[0103] The collected culture medium was centrifuged at 3,600 rpm for 30 minutes, and the supernatant was transferred to a centrifuge tube with a cellulose membrane and concentrated by centrifugation at 3,600 rpm for 20 minutes. The conditioned medium that passed through the membrane after centrifugation was discarded, and culture medium equivalent to the discarded conditioned medium was added and concentrated. Centrifugation was continued until the concentrated culture medium was less than 1 ml, and the completed concentrated culture medium was quantified by Bradford assay. The concentrated culture medium was mixed with DMEM medium to a final concentration of 1 mg / ml.

[0104] A membrane coated with antibodies capable of detecting 507 cytokines (Cytokine Array Kit, RayBiotech, USA) was treated with blocking buffer and incubated for 30 minutes. The blocking buffer was removed from the membrane, and the membrane was replaced with concentrated culture medium and incubated overnight under refrigerated conditions. The membrane was washed seven times with washing buffer. HRP-conjugated streptavidin solution was added to the membrane and incubated at room temperature for 2 hours. After removing the HRP-conjugated streptavidin solution, the membrane was washed seven times with washing buffer. After washing, the membrane was wetted with enhanced chemiluminescence (ECL) reagent and cytokine expression was confirmed using an imager.

[0105] The results of a heat map comparison of cytokine expression in nerve regeneration-promoting cells are shown in Figure 12. As shown in Figure 12, it was confirmed that the expression patterns of nerve regeneration-promoting cells and tonsil-derived mesenchymal stem cells were different from each other.

[0106] As a result of comparing cytokine expression in mesenchymal stem cells and nerve regeneration-promoting cells derived from T-MSC-1-1 and T-MSC-1-2, the cytokines whose expression was increased were as shown in FIG.

[0107] - More than 1.5-fold: Angiopoietin-1, Angiopoietin-4, BIK, BMPR-IA / ALK-3, CCL14 / HCC-1 / HCC-3, CCR1, EN-RAGE, Eotaxin-3 / CCL26, FGF R4, FGF-10 / KGF-2, FGF-19, FGF-21, Flt-3 Ligand, Follistatin-like 1, GASP-1 / WFIKKNRP, GCP-2 / CXCL6, GFR alpha-3, GREMLIN, GRO-a, HGF, HRG-beta 1, I-309, ICAM-1, IFN-alpha / beta R2, IGFBP-2, IGF-I, IL-4, IL-5 R alpha, IL-10 R beta, IL-1 2R beta 1, IL-13 R alpha 2, IL-​​​- 2x or more: BIK, GRO-a, HGF, MCP-4 / CCL13, uPA In summary, the present inventors prepared nerve regeneration-promoting cells from tonsillar and adipose-derived mesenchymal stem cells and confirmed their expression patterns by CD marker analysis. Furthermore, they confirmed the nerve regeneration effects of the nerve regeneration-promoting cells. This means that cells with nerve regeneration effects can be prepared from tonsillar tissue that would otherwise be discarded as medical waste, and the nerve regeneration-promoting cells of the present invention can be used in a variety of ways in the field of nerve regeneration.

[0108] The above describes an embodiment of the present invention, but a person having ordinary knowledge in the field of technology can modify and change the present invention in various ways by adding, changing, deleting or adding components, etc., within the scope of the idea of ​​the present invention described in the claims, and it can be said that these modifications and changes are also included in the scope of the present invention. [Brief explanation of the drawings]

[0109] [Figure 1] 1 shows photographs taken on a daily basis of the results when T-MSC-1-1, which are tonsil-derived mesenchymal stem cells, were induced to become nerve regeneration-promoting cells. [Figure 2] 1 shows the results of CD marker expression in the nerve regeneration-promoting cells according to the present invention, as determined by CD marker screening, shown in a heat map. [Figure 3] These are the results of screening for CD markers that have different expression levels in nerve regeneration-promoting cells compared to tonsil-derived mesenchymal stem cells. Figure 3A shows the results of comparing CD markers with increased expression levels compared to tonsil-derived mesenchymal stem cells, and Figure 3B shows the results of comparing CD markers with decreased expression levels compared to tonsil-derived mesenchymal stem cells. [Figure 4] This is the result of comparing the expression patterns of CD markers whose expression was increased and decreased in nerve regeneration-promoting cells compared to tonsil-derived mesenchymal stem cells. [Figure 5] 1 is a histogram showing the results of screening CD markers whose expression was commonly increased or decreased in nerve regeneration-promoting cells. [Figure 6] The expression patterns of markers CD121a, CD106, and CD112, which are commonly increased in nerve regeneration-promoting cells, are compared with those of tonsil-derived mesenchymal stem cells. [Figure 7] The expression patterns of markers CD26 and CD141, which are commonly decreased in nerve regeneration-promoting cells, are compared with those of tonsil-derived mesenchymal stem cells. [Figure 8] FIG. 1 shows a heat map of the results of comparing the expression of CD markers in order to compare the CD marker expression patterns of tonsil-derived mesenchymal stem cells (T-MSCs) and nerve regeneration-promoting cells (NRPCs). [Figure 9] These are the results of a neurite outgrowth assay performed to measure and compare the growth of neurites from nerve regeneration-promoting cells. [Figure 10] This result confirmed that the process of co-culturing candidate cells with dorsal root ganglia resulted in myelination in some cells morphologically. [Figure 11] These are the results of flow cytometry analysis using individual antibodies for up- and down-regulated CD markers selected in CD screening. [Figure 12] The results of cytokine array analysis of T-MSCs and NRPCs are shown in a heat map (left), and the results of dividing the cytokines that were commonly increased in NRPCs compared to T-MSCs by ratio (right) (fold change: NRPC1-1 / T-MSC1-1, NRPC1-2 / T-MSC1-2).

Claims

1. A method for screening cells having nerve regeneration activity from cells differentiated from tonsil-derived mesenchymal stem cells, comprising: (i) selecting cells from the differentiated cells in which markers including CD121a, CD106, and CD112 are up-regulated compared to mesenchymal stem cells before differentiation; and (ii) selecting cells from the differentiated cells in which markers including CD26 and CD141 are down-regulated compared to mesenchymal stem cells before differentiation. (a) the differentiated cells are formed by culturing tonsil-derived mesenchymal stem cells to form neurospheres, which are then differentiated; (b) a culture medium for differentiating neurospheres into cells having nerve regeneration activity, comprising (1) GlutaMAX-containing DMEM / F12, and (2) three or more selected from the group consisting of 5-20% FBS, 5-20 ng / ml bFGF, 100-400 μM butylhydroxyanisole, 5-40 μM forskolin, 0.1-10% N2 supplement, 1-100 ng / ml brain-derived nerve growth factor, 1-100 ng / ml nerve growth factor, 0.01-1 ng / ml sonic hedgehog, 1-10 ng / ml PDGF-AA, and 50-300 ng / ml heregulin beta 1; (c) the expression of the marker CD121a is upregulated by 30% or more compared to said tonsil-derived mesenchymal stem cells; (d) the expression of the marker CD106 is upregulated by 5% or more compared to said tonsil-derived mesenchymal stem cells; (e) the expression of the marker CD112 is upregulated by 10% or more compared to the tonsil-derived mesenchymal stem cells; (f) the expression of the marker CD26 is downregulated by 5% or more compared to said tonsil-derived mesenchymal stem cells; and (g) the expression of the marker CD141 is downregulated by 5% or more compared to the tonsil-derived mesenchymal stem cells; Screening methods.

2. The cell screening method according to claim 1, wherein the nerve regeneration activity includes myelination of peripheral nerves.

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