Method for screening neuronal regeneration-promoting cells that have neuronal regeneration activity

Abstract

The present invention relates to a method for selecting neuronal regeneration-promoting cells derived from mesenchymal stem cells, with neuronal regenerative activity, and to a pharmaceutical composition comprising such cells. The neuronal regeneration-promoting cells of the present invention are completely different from stem cells with respect to the expression pattern of a CD marker and exhibit an excellent neuronal regenerative effect, and therefore may have various applications in the prophylactic or therapeutic field of neuropathy.

Description

Method for screening neuronal regeneration-promoting cells that have neuronal regeneration activity Technical field The present description refers to a method for screening neuronal regeneration-promoting cells derived from stem cells that have neuronal regeneration activity and a pharmaceutical composition for preventing or treating a neurological disease, containing the neuronal regeneration-promoting cells. Previous technique Mesenchymal stem cells (MSCs) are widely used in the development of cell therapy agents because they can differentiate into a variety of cell types in response to specific stimulation and are free from the tumorigenicity of induced pluripotent stem cells and the ethical issues surrounding the use of embryonic stem cells. Mesenchymal stem cells are adult stem cells and are commonly isolated from adult tissues such as adipose tissue, umbilical cord blood, and bone marrow. Methods for isolating these tissues are invasive, painful, and cannot yield large numbers of stem cells. Technical problem The inventors of the present description have derived a method for screening neuronal regeneration-promoting cells that exhibit neuronal regeneration effect from among several differentiated mesenchymal stem cell cells by analyzing specific CD markers. This description aims to provide a method for screening neuronal regeneration-promoting cells derived from mesenchymal stem cells that have neuronal regeneration activity. This description also aims to provide neuronal regeneration-promoting cells screened using the screening method. The present description also aims to provide a pharmaceutical composition for preventing or treating a neurological disease, containing neuronal regeneration-promoting cells as an active ingredient. Other purposes and advantages of the present description will be made more evident through the following detailed description, claims, and figures. Technical solution In one respect, the present description provides a method for screening neuronal regeneration-promoting cells derived from mesenchymal stem cells that have neuronal regeneration activity, which includes: i) a stage of preparing differentiated cells from mesenchymal stem cells; and ii) a cell screening step in which one or more markers selected from a group consisting of CD121a, CD106 and CD112 are upregulated among differentiated cells from step i) compared to mesenchymal stem cells prior to differentiation. In another aspect, the present description provides a method for screening neuronal regeneration-promoting cells derived from mesenchymal stem cells that have neuronal regeneration activity, which includes: i) a stage of preparing differentiated cells from mesenchymal stem cells; and ii) a cell screening step in which one or more markers selected from a group consisting of CD26 and CD141 are downregulated from among differentiated cells from step i) compared to mesenchymal stem cells prior to differentiation. The inventors of the present description differentiated mesenchymal stem cells derived from various sources and investigated the expression pattern of several markers in the various differentiated cells. As a result, it was surprisingly confirmed that cells differentiated into neuronal regeneration-promoting cells show a common trend in the expression pattern of specific markers (e.g., CD markers such as CD121a, CD106, and CD112). In the present description, the term "neuronal regeneration-promoting cell", or "NRPC", refers to a cell differentiated from a mesenchymal stem cell, which has a neuronal regeneration effect (e.g., an effect of promoting neuronal regeneration directly or indirectly in terms of structure or function by myelination of damaged peripheral nerves or secretion of cytokines necessary for neuronal regeneration). According to a specific illustrative modality of the present description, the screening method includes: i) a step of preparing differentiated cells from mesenchymal stem cells; (ii) a cell screening step in which one or more markers selected from a group consisting of CD121a, CD106 and CD112 are upregulated among differentiated cells from step (i) compared to pre-differentiated mesenchymal stem cells; and iii) a cell screening step in which one or more markers selected from a group consisting of CD26 and CD141 are downregulated from among differentiated cells of step i) compared to mesenchymal stem cells prior to differentiation. According to a specific illustrative modality of the present description, the screening method includes: i) a step of preparing differentiated cells from mesenchymal stem cells; (ii) a cell screening step in which one or more markers selected from a group consisting of CD26 and CD141 are downregulated among differentiated cells from step (i) compared to mesenchymal stem cells before differentiation; and iii) a cell screening step in which one or more markers selected from a group consisting of CD121a, CD106 and CD112 are upregulated from among differentiated cells of step i) compared to mesenchymal stem cells prior to differentiation. In this description, the term "stem cell" refers to a cell that can replicate itself and simultaneously has the capacity to differentiate into two or more cell types. Stem cells include adult stem cells, pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells. Specifically, they can be mesenchymal stem cells. In this description, the term "mesenchymal stem cell" refers to an undifferentiated stem cell isolated from the tissue of a human or mammal. Mesenchymal stem cells can be derived from various tissues. Specifically, they can be derived from one or more tissues selected from a group consisting of tonsils, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, chorion, decidua, and placenta. The techniques for isolating stem cells from each tissue are well established in the field. According to a specific illustrative modality of the present description, the mesenchymal stem cell is derived from the tonsils or fat. In one example of the present description, it was verified that it is most preferred to use mesenchymal stem cells derived from tonsils or fat. In this description, the term "CD" or "differentiation group" refers to a surface molecular structure present on the cell surface. Since some cell populations share the same CD molecules, they are used to distinguish cell populations (i.e., as markers). Cells of the same lineage have the same CD molecules, but even within the same cell population, they have different CD molecules depending on the stage of differentiation or activation. Therefore, they are useful for identifying cell lineage, differentiation, activation, and other characteristics. In an example from the present description, it was verified by comparing the expression pattern of CD molecules in neuronal regeneration-promoting cells derived from neuronally regenerating stem cells of the present description and mesenchymal stem cells that neuronal regeneration-promoting cells, according to the present description, are different from mesenchymal stem cells. In the present description, CD10, CD39, CD106, CD112, CD121a, CD338, etc., whose expression is upregulated compared to mesenchymal stem cells, or CD26, CD54, CD126, CD141, etc., whose expression is downregulated, can be used as markers of neuronal regeneration-promoting cell differentiation. According to a specific illustrative modality of the present description, the differentiated cells of stage i) are differentiated from neurospheres formed by culturing mesenchymal stem cells. According to a specific illustrative modality of the present description, neuronal regeneration activity includes myelination of peripheral nerves. In this description, the term "myelination" refers to the process by which myelin surrounds the axons of peripheral nerves to increase the speed at which a stimulus is delivered. Damaged peripheral nerves are repaired (i.e., regenerated) through myelination. In an example from the present description, some of the candidate cells screened using the screening method described herein were cytomorphologically myelinized by co-culture with dorsal root ganglia. In another aspect, the present description provides neuronal regeneration-promoting cells screened using the screening method described above. According to a specific illustrative modality of the present description, neuronal regeneration-promoting cells have the following characteristics: a) the expression of the markers CD121a, CD106 and CD112 is upregulated compared to mesenchymal stem cells before differentiation; and b) The expression of the markers CD26 and CD141 is negatively regulated compared to mesenchymal stem cells before differentiation. In neuronal regeneration-promoting cells, the expression of the CD121a marker is upregulated by 30% or more, more specifically by 40% or more, compared to mesenchymal stem cells before differentiation. In one example of the present description, the expression of the CD121a marker was found to be upregulated by 94% in T-MSC-1-1 derived neuronal regeneration promoter cells, by 71% in T-MSC-1-2 derived neuronal regeneration promoter cells, by 51% in T-MSC-1-3 derived neuronal regeneration promoter cells, and by 48% in T-MSC-1-4 derived neuronal regeneration promoter cells, by an average of 66% or more, compared to mesenchymal stem cells before differentiation. In neuronal regeneration-promoting cells, the expression of the CD106 marker is upregulated by 5% or more, more specifically by 10% or more, compared to mesenchymal stem cells before differentiation. In one example of the present description, the expression of the CD106 marker was found to be upregulated by 30% in neuronal regeneration promoter cells derived from T-MSC-1-1, by 11% in neuronal regeneration promoter cells derived from T-MSC-1-2, by 16% in neuronal regeneration promoter cells derived from T-MSC-1-3, and by 13% in neuronal regeneration promoter cells derived from T-MSC-1-4, by 17% or more on average, compared to mesenchymal stem cells before differentiation. In neuronal regeneration-promoting cells, the expression of the CD112 marker is upregulated by 10% or more, more specifically by 15% or more, compared to mesenchymal stem cells before differentiation. In one example of the present description, the expression of the CD112 marker was found to be upregulated by 49% in T-MSC-1-1 derived neuronal regeneration promoter cells, by 25% in T-MSC-1-2 derived neuronal regeneration promoter cells, by 30% in T-MSC-1-3 derived neuronal regeneration promoter cells, and by 19% in T-MSC-1-4 derived neuronal regeneration promoter cells, by 30% or more on average, compared to mesenchymal stem cells before differentiation. According to a specific illustrative modality of the present description, in neuronal regeneration-promoting cells, the expression of the CD26 marker is negatively regulated compared to mesenchymal stem cells before differentiation. In neuronal regeneration-promoting cells, the expression of the CD26 marker is specifically downregulated by 5% or more, more specifically by 8% or more, compared to mesenchymal stem cells before differentiation. In one example from the present description, the expression of the CD26 marker is downregulated by 9% in T-MSC-1-1 derived neuronal regeneration promoter cells, by 11% in T-MSC-1-2 derived neuronal regeneration promoter cells, by 27% in T-MSC-1-3 derived neuronal regeneration promoter cells, and by 16% in T-MSC-1-4 derived neuronal regeneration promoter cells, by 16% or more on average, compared to mesenchymal stem cells before differentiation. In neuronal regeneration-promoting cells, the expression of the CD141 marker is specifically downregulated by 5% or more, more specifically by 8% or more, compared to mesenchymal stem cells before differentiation. In one example from the present description, the expression of the CD141 marker is downregulated by 9% in T-MSC-1-1 derived neuronal regeneration promoter cells, by 20% in T-MSC-1-2 derived neuronal regeneration promoter cells, by 16% in T-MSC-1-3 derived neuronal regeneration promoter cells, and by 38% in T-MSC-1-4 derived neuronal regeneration promoter cells, by 20% or more on average, compared to mesenchymal stem cells before differentiation. In another aspect, the present description provides a pharmaceutical composition for preventing or treating a neurological disease, containing neuronal regeneration-promoting cells as an active ingredient. In another aspect, the present description provides a method for treating a neurological disease, which includes a step of administering an effective amount of neuronal regeneration-promoting cells to a subject. In another aspect, the present description provides a use of neuronal regeneration-promoting cells in therapy. In the present description, the term "neurological disease" refers to a disease caused by damage to nerve tissue due to intrinsic factors such as heredity, aging, etc., or extrinsic factors such as trauma, etc. According to a specific illustrative modality of the present description, neurological disease is one or more diseases selected from a group consisting of Charcot-Marie-Tooth neuropathy, diabetic peripheral neuropathy, spinal cord injury, amyotrophic lateral sclerosis, carpal tunnel syndrome, infantile paralysis, leprosy, muscular dystrophy, polymyositis, and myasthenia gravis. In the present description, the term "subject" refers to an individual requiring administration of the neuronal regeneration-promoting composition or cells of the present description, and includes mammals, birds, reptiles, amphibians, fish, etc. without limitation. In this description, "prevention" refers to any action of inhibiting or delaying a neurological disease by administering the composition according to this description. "Treatment" refers to any action of improving or favorably altering the symptoms of a neurological disease by administering the composition according to this description. According to a specific illustrative modality in this description, the pharmaceutical composition herein contains a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of the present description can be prepared in a single-dose or multiple-dose formulation by using a pharmaceutically acceptable carrier and / or excipient according to a method that can be readily carried out by those having ordinary knowledge in the art to which the present description pertains. The pharmaceutical composition described herein can be prepared in various formulations using common methods. For example, it can be prepared as an oral formulation such as a powder, granule, tablet, capsule, suspension, emulsion, syrup, etc., and also as a formulation for external application, such as a suppository or a sterile injection solution. The composition described herein may contain one or more known active ingredients that have a preventive or therapeutic effect on a neurological disease, along with neuronal regeneration-promoting cells derived from stem cells that have neuronal regeneration activity. The pharmaceutical composition described herein can be administered orally or parenterally.Specifically, it can be administered parenterally, for example, by intravenous injection, transdermal administration, subcutaneous injection, intramuscular injection, intravitreal injection, subretinal injection, suprachoroidal injection, eye drop administration, intracerebroventricular injection, intrathecal injection, intra-amniotic injection, intra-arterial injection, intra-articular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisternal injection, intracoronary injection, intracranial injection, intradural injection, epidural 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 injection, intratumoral injection, topical injection, etc. Parenteral formulations include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized formulation, and a suppository. For the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, an injectable ester such as ethyl oleate, etc., may be used. For the suppository base, witepsol, macrogol, Tween 61, cocoa butter, lauryl butter, glycerogelatin, etc., may be used. The dosage for administration of the pharmaceutical composition described herein may vary depending on several factors such as the method of formulation, the method of administration, the time of administration, the route of administration, the response to be achieved with the administration of the pharmaceutical composition and the extent thereof, the age, body weight, general health status, pathological condition or severity, sex, diet, and excretion rate of a subject to whom the pharmaceutical composition is administered, and other drugs or ingredients used together and similar factors well known in the medical field, and an effective administration dose for the desired treatment can be easily determined and prescribed by those with ordinary knowledge of the technique. The route of administration and the method of administration of the pharmaceutical composition described herein may be independent of each other and are not especially limited as long as the pharmaceutical composition can reach the target site. Advantageous Effects The features and advantages of this description can be summarized as follows: (i) The present description provides a method for screening neuronal regeneration-promoting cells derived from mesenchymal stem cells that have neuronal regeneration activity and a pharmaceutical composition containing the neuronal regeneration-promoting cells. (ii) The neuronal regeneration-promoting cells described herein are completely different from stem cells in terms of the expression pattern of a CD marker and exhibit an excellent neuronal regeneration effect. Consequently, they can be applied in various fields to prevent or treat neurological diseases. Brief description of the figures Figure 1 shows images of neuronal regeneration-promoting cells induced from tonsil-derived T-MSC-1-1 mesenchymal stem cells. Figure 2 shows heat maps visualizing the expression of CD markers in neuronal regeneration-promoting cells according to the present description through CD marker screening. Figures 3a and 3b show the results of CD marker screening, revealing the difference in expression levels between neuronal regeneration-promoting cells and 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, while Figure 3b shows the results of comparing CD markers with decreased expression levels compared to tonsil-derived mesenchymal stem cells. Figure 4 shows the result of comparing the expression pattern of CD markers whose expression has increased and CD markers whose expression has decreased in neuronal regeneration promoter cells compared to mesenchymal stem cells derived from the tonsils. Figure 5 shows a screening result for CD markers whose expression has increased or decreased in neuronal regeneration-promoting cells. Figure 6 shows the result of comparing the expression pattern of the markers CD121a, CD106 and CD112 whose expression has increased in neuronal regeneration promoter cells compared to mesenchymal stem cells derived from the tonsils. Figure 7 shows the result of comparing the expression pattern of the markers CD26 and CD141, whose expression has decreased in neuronal regeneration promoter cells compared to mesenchymal stem cells derived from the tonsils. Figure 8 shows the result of comparing the expression pattern of CD markers in tonsil-derived mesenchymal stem cells (T-MSCs) and neuronal regeneration promoter cells (NRPCs) using heat maps. Figure 9 shows a result of performing the neurite growth assay to compare neurite growth from neuronal regeneration-promoting cells. Figure 10 shows that myelination was achieved cytomorphologically in some of the candidate cells co-cultured with dorsal root ganglia. Figure 11 shows a result of performing flow cytometry for positively regulated and negatively regulated CD markers screened by CD screening using individual antibodies. Figure 12 shows the visualization results of the cytokine matrix assay of T-MSCs and NRPCs using heat maps (left) and the proportion of cytokines increased in NRPCs compared to T-MSCs (right) (change in times: NRPC 1-1 / T-MSC 1-1, NRPC 1-2 / T-MSC 1-2). Optimal mode The following description will be illustrated in more detail through examples. These examples are provided solely to illustrate the present description more specifically, and it will be obvious to those with ordinary knowledge of the art that the scope of this description is not limited by them. Examples Example 1. Preparation of mesenchymal stem cells 1-1. Isolation and culture of mesenchymal stem cells derived from tonsils Tonsil tissue from multiple donors acquired from the Ewha Womans University School of Medicine was placed in a tube containing 10 ml of DPBS (Dulbecco's phosphate-buffered saline) supplemented with 20 µg / ml gentamicin, centrifuged at 1500 rpm for 5 minutes, and then washed twice. The washed tonsils were then sectioned using sterile scissors. To isolate tonsil-derived mesenchymal stem cells from tonsil problems, after adding an enzyme reaction solution of the same weight, the tonsil problems were incubated in a shaking incubator at 37 °C and 200 rpm for 60 minutes. The composition of the enzyme reaction solution is described in Table 1. Table 1 After adding 5% FBS (fetal bovine serum) to the culture, the mixture was centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the remaining pellets were resuspended in 30 ml of DPBS and then centrifuged again at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the remaining pellets were 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 on the filter were washed with 20 ml of DPBS and then centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the cells were incubated in a constant-temperature water bath at 37 °C for 5 minutes after adding ACK lysis buffer. After adding DPBS to the suspension, centrifugation was performed at 1500 rpm for 5 minutes.After centrifugation, the supernatant was discarded, and the remaining pellets were resuspended in high-glucose DMEM (10% FBS, 20 µg / ml gentamicin) to prepare a cell suspension. The number of cells in the prepared cell suspension was then counted. The cell suspension was plated into a T175 flask and incubated at 37 °C with 5% CO₂ in a CO₂ incubator. 1-2. Isolation and culture of mesenchymal stem cells derived from adipose tissue Adipose tissue-derived mesenchymal stem cells were purchased from Lonza (human adipose tissue-derived stem cells, Cat#PT-5006, Lonza, Switzerland). The adipose tissue-derived mesenchymal stem cells were cultured using a medium provided by Lonza (Bulletkit ADSD, Cat#PT-4505). Example 2. Formation of neurospheres Neurospheres were formed by culturing the mesenchymal stem cells from Example 1. Specifically, the mesenchymal stem cells were subcultured to 4–7 passages. After removing the culture medium, the mesenchymal stem cells were washed with DPBS. After treating the washed cells with Tr and pLE, the harvested cells were counted. After centrifuging the harvested cells and removing the supernatant, they were resuspended in a neurosphere-forming medium. The composition of the neurosphere-forming medium is described in Table 2. Table 2 The cells resuspended in the neurosphere (1 x 10⁶ cells) were seeded in an ultra-low junction dish (60 mm). The seeded cells were cultured for 3 days at 37 °C and 5% CO₂. After 3 days of culture, the neurospheres formed in the dish were collected in a 15 ml tube. After centrifuging the collected cells and removing the supernatant, a neurosphere suspension was prepared by adding fresh neurosphere-forming medium. The neurosphere suspension was transferred to an ultra-low junction dish, and the neurospheres were cultured for 4 days at 37 °C and 5% CO₂. Example 3. Differentiation of neuronal regeneration-promoting cells (NRPCs) into candidate cells using neurospheres The neurospheres formed in Example 2 were finely ground using a 23-26 G syringe needle. The ground neurospheres were transferred to a 15 ml tube using a pipette and then centrifuged. After removing the supernatant, the ground neurospheres were resuspended by adding a neuronal regeneration-promoting cell induction medium to the tube. Several cell induction media were prepared that promote neuronal regeneration by combining three or more of the following: 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 supplement (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).) , 8) 0.01-1 ng / ml of sonic hedgehog (SHH, R & D Systems, USA) , 9) 1-10 ng / ml of PDGF-AA (platelet-derived growth factor-AA, Peprotech, USA) and 10) 50-300 ng / ml of heregulin-beta1, Peprotech, USA) in DMEM / F12 containing GlutaMAX. The neurospheres resuspended in the various media were seeded in a T175 flask coated with laminin (2 µg / ml). The seeded neurospheres were cultured for 8-10 days while the neuronal regeneration-promoting cell induction medium was exchanged at 3-day intervals (Figure 1). Example 4. First screening of candidate cells of neuronal regeneration promoter cells through confirmation of myelination of peripheral nerves We investigated whether the neuronal regeneration-promoting cell candidates prepared in Example 3 have the capacity to myelinate peripheral nerves. Specifically, the differentiated neuronal regeneration-promoting cell candidates were co-cultured with dorsal root ganglia (DRGs), and we investigated whether myelination occurred. Rat dorsal root ganglion (DRG) cells were purchased from Lonza (rat dorsal root ganglion cells, Cat# R-DRG-505, Lonza, Switzerland). Candidate cells were co-cultured with the purchased dorsal root ganglia. The DRG cells were cultured using a culture medium provided by Lonza (primary neuronal growth medium (PNGM) pellet kit, Cat. No. CC-4461). The culture medium was exchanged every 3 days. As a result of co-culturing the candidate cells with the dorsal root ganglia, it was confirmed that myelination was achieved cytomorphologically in some of the cells (Figure 10). Example 5. Second screening of neuronal regeneration promoter cells by analyzing the expression of CD markers The expression of a total of 242 CD markers was analyzed in T-MSC-1-1 (mygdala-derived mesenchymal stem cells 1), T-MSC-1-2 (mygdala-derived mesenchymal stem cells 2), T-MSC-1-3 (mygdala-derived mesenchymal stem cells 3), and T-MSC-1-4 (mygdala-derived mesenchymal stem cells 4), where myelination was cytomorphologically confirmed between the neuronal regeneration promoter cell candidates in Example 4, and the neuronal regeneration promoter cells differentiated from these. For CD marker analysis, 3 x 10⁷ target cells were collected. The target cells were washed with DPBS and then centrifuged at 2000 rpm for 5 minutes. After removing the supernatant and washing once with DPBS, centrifugation was performed, and the remaining pellets were resuspended in 30 ml of FACS buffer. 100 µl of the cell suspension (1 x 10⁵ cells) was seeded into each well of a 96-well round-bottom plate. Then, 10 µl of CD marker primary antibodies were added to each well of the 96-well plate. After incubation for 30 minutes on ice in the dark, each well was washed with 100 µl of FACS buffer and then centrifuged at 300 g for 5 minutes. After removing the supernatant and adding 200 µl of FACS buffer to each well, centrifugation was performed at 300 g for 5 minutes. Secondary antibodies were prepared in FACS buffer at a ratio of 1:200 (1.25 µg / ml).After centrifugation, the supernatant was discarded, and 100 µl of the prepared secondary antibodies were added to each well. After reaction for 20–30 minutes on ice in the dark, each well was washed with 100 µl of FACS buffer and then centrifuged at 300 g for 5 minutes. After removing the supernatant, the target cells were washed by adding 200 µl of FACS buffer to each well. The washing procedure was repeated twice. After washing, the cells were resuspended by adding 200 µl of FACS buffer to each well, and CD marker expression in the target cells was investigated by flow cytometry or FACS (fluorescence-activated cell sorting). The result of comparing the expression of CD markers in neuronal regeneration-induced promoter cells using heat maps is shown in Figure 2. As shown in Figure 2, neuronal regeneration-promoting cells (NRPCs) and mesenchymal stem cells (MSCs) showed similar CD marker expression patterns, but showed differences in the expression pattern of some markers. The expression patterns of CD markers in mesenchymal stem cells (MSCs) T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 and neuronal regeneration-promoting cells (NRPCs) were compared to select CD markers whose expression has increased or decreased as markers of neuronal regeneration-promoting cell differentiation. The CD markers whose expression has increased or decreased are shown in Figure 3. As shown in Figure 3a, the CD markers whose expression has increased in neuronal regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 (in at least three of the four NRPCs) compared to tonsil-derived mesenchymal stem cells were CD10, CD39, CD106, CD112, CD121a, CD338, etc. And, as shown in Figure 3b, the CD markers whose expression has decreased (in at least three of the four NRPCs) were CD26, CD54, CD126, CD141, etc. The result of comparing the increase and decrease of CD markers in neuronal regeneration-promoting cells derived from the amygdalae is shown in Figure 4. As shown in Figure 4, the expression of 12 CD markers increased and the expression of 9 CD markers decreased in T-MSC-1-1-derived neuronal regeneration promoter cells. In T-MSC-1-2-derived neuronal regeneration promoter cells, the expression of 8 CD markers increased and the expression of 9 CD markers decreased. In T-MSC-1-3-derived neuronal regeneration promoter cells, the expression of 40 CD markers increased and the expression of 3 CD markers decreased. In T-MSC-1-4-derived neuronal regeneration promoter cells, the expression of 17 CD markers increased and the expression of 6 CD markers decreased. Based on these results, CD markers whose expression was commonly increased or decreased in T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4-derived neuronal regeneration promoter cells were screened. The screened markers are as follows: - CD markers whose expression has commonly increased: CD106, CD112 and CD121a. - CD markers whose expression has commonly decreased: CD26 and CD141. The pattern of CD markers whose expression has increased or decreased was commonly observed in an identical manner also in neuronal regeneration promoter cells differentiated from adipose tissue-derived mesenchymal stem cells of Example 1-2. The result of the CD projection of CD markers whose expression has increased or decreased in neuronal regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 is shown in Figure 5. As shown in Figure 5, the expression level of CD markers whose expression is commonly increased in neuronal regeneration-promoting cells—namely, CD121a, CD106, and CD112—increased by 10% or more after differentiation. Meanwhile, the expression level of markers whose expression is commonly decreased—namely, CD26 and CD141—decreased by approximately 9% or more. This result suggests that the commonly altered expression markers CD121a, CD106, CD112, CD26, and CD141 can be used as differentiation markers for neuronal regeneration-promoting cells. In particular, CD121a, CD106, and CD112 can be used as representative differentiation markers. 6-1. Comparison of the expression of the co-expression markers CD121a, CD106 and CD112 The expression of the markers CD121a, CD106, and CD112 is commonly increased in neuronal regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4 by 10% or more compared to mesenchymal stem cells, which is the most prominent characteristic of neuronal regeneration-promoting cells. The results of the comparison of CD121a, CD106, and CD112 expression in neuronal 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 Figure 6. As shown in Figure 6, the expression of the markers CD121a, CD106, and CD112 was markedly increased in tonsil-derived mesenchymal stem cells (T-MSCs) compared to neuronal mesenchymal stem cells (NRPCs). 6-2. Comparison of the expression of the co-expression markers CD26 and CD141 The expression of the CD26 and CD141 markers was commonly decreased in neuronal regeneration-promoting cells derived from T-MSC-1-1, T-MSC-1-2, T-MSC-1-3, and T-MSC-1-4. The results of the comparison of CD marker expression in neuronal 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 Figure 7. As shown in Figure 7, the expression of CD26 and CD141 decreased in neuronal regeneration promoter cells compared to tonsil-derived mesenchymal stem cells. 6-3. Comparison of the expression pattern of CD markers To compare the expression pattern of CD markers in tonsil-derived mesenchymal stem cells and neuronal regeneration promoter cells, heat maps were constructed based on the results of comparing the expression of co-expressed CD markers in Examples 6-1 and 6-2. The result is shown in Figure 8. As shown in Figure 8, neuronal regeneration-promoting cells showed different expression of co-expression markers of tonsil-derived mesenchymal stem cells. 6-4. Average expression of co-expression CD markers To investigate whether the expression pattern of CD markers in tonsil-derived mesenchymal stem cells and neuronal regeneration promoter cells is maintained after cell freezing, the expression of co-expressed CD markers was confirmed in live, frozen, and thawed cells. The results are shown in Figure 11. As shown in Figure 11, the expression of CD106, CD121a, and CD112 increased, while the expression of CD26 and CD141 decreased, in neuronal regeneration-promoting cells compared to tonsil-derived mesenchymal stem cells, even after freezing. Neuronal regeneration-promoting cells exhibited different expression of co-expression markers compared to tonsil-derived mesenchymal stem cells, regardless of freezing. Example 7. Neurite growth effect of neuronal regeneration-promoting cells The neurite (or neuronal process), which projects from the cell body of a neuron, is known to be involved in the transport of substances necessary for axon growth and regeneration, such as neurotransmitters, nerve growth factors, etc. (L McKerracher et al., Spinal Cord Repair: Strategies to Promote Axon Regeneration, Neurobiol Dis, 2001). A neurite growth assay was performed to compare neurite growth in the neuronal regeneration-promoting cells described herein. N1E-115 cells (mouse neuroblastoma cells, ATCC, USA) were cultured and seeded onto a microporous filter (neurite growth assay kit, Millipore, USA). The seeded cells were cultured for 48 hours in a culture medium from which neuronal regeneration-promoting cells or stem cells were collected. Absorbance was measured after staining the neurites projected through a fine porous filter. The neurite growth assay confirmed that culturing neuronal regeneration-promoting cells regulates or stimulates neurite (axon) growth in N1E-115 cells (mouse neuroblastoma). Neurite growth in cultured N1E-115 cells was compared with that of T-MSC-1-2-derived neuronal regeneration-promoting cells. The results are shown in Figure 9 (NRPC: T-MSC-1-2-derived neuronal regeneration-promoting cells, T-MSC: T-MSC-1-2, Negative control: negative control group, Positive control: positive control group). A large number of neurites were observed in the neuronal regeneration-promoting cells compared with the amygdala-derived stem cells, and absorbance was also increased in the neuronal regeneration-promoting cells compared with the amygdala-derived stem cells (negative control: a porous filter (membrane insert provided with a neurite growth assay kit) was coated with BSA and the N1E-115 cells were cultured in DMEM (+ 20 µg / ml gentamicin)).Positive control: a porous filter was coated with laminin and N1E-115 cells were cultured in DMEM (+ 20 µg / ml gentamicin + 1 mg / ml BSA). NRPC and T-MSC groups: a porous filter was coated with BSA and N1E-115 cells were cultured in an NRPC or T-MSC culture. Example 8. Cytokine matrix assay of neuronal regeneration-promoting cells The expression of 507 cytokines was analyzed in T-MSC-1-1 and T-MSC-1-2, which showed the most prominent myelination in Example 4, and in the neuronal regeneration-promoting cells differentiated from these. Target cells were cultured for cytokine analysis. Target cells were seeded in a flask and cultured for 3–4 days. When the target cells filled 80% or more of the flask's surface area, the culture medium was removed, and the target cells were washed twice with DPBS. After washing, the culture medium was replaced with DMEM (Dulbecco's phosphate-buffered saline) that did not contain FBS (fetal bovine serum), cytokines, etc., to eliminate the effect of cytokines. The target cell culture was harvested after 30 hours of culture. The collected culture was centrifuged at 3600 rpm for 30 minutes. The supernatant was transferred to a centrifuge tube fitted with a cellulose membrane and concentrated by centrifugation at 3600 rpm for 20 minutes. After centrifugation, the conditioned medium that passed through the separating membrane was discarded, and an equal amount of culture was added. Centrifugation continued until the volume of the concentrated culture was reduced to 1 ml or less, and the concentrated culture was quantified using the Bradford assay. The concentrated culture was adjusted to a final concentration of 1 mg / ml by mixing with DMEM. A membrane coated with antibodies capable of detecting 507 cytokines (Cytokine Array Kit, RayBiotech, USA) was reacted for 30 minutes by treatment with a blocking buffer. After removing the remaining blocking buffer from the membrane and replacing it with the concentrated culture, the membrane was reacted overnight in a refrigerator. The membrane was washed seven times with a wash buffer. After adding a solution of HRP-conjugated streptavidin, the membrane was reacted at room temperature for 2 hours. After removing the HRP-conjugated streptavidin solution, the membrane was washed seven times with a wash buffer. After washing, the membrane was soaked with an ECL (enhanced chemiluminescence) reagent, and cytokine expression was confirmed using an imaging device. The results of comparing cytokine expression in neuronal regeneration promoter cells using heat maps are shown in Figure 12. As shown in Figure 12, neuronal regeneration promoter cells and tonsil-derived mesenchymal stem cells showed different expression patterns. The cytokines whose expression has increased in mesenchymal stem cells and neuronal regeneration-promoting cells derived from T-MSC-1-1 and T-MSC-1-2 are shown in Figure 12. - 1, 5 veces o más: angiopoyetina-1, angiopoyetina-4, BIK, BMPR-IA / ALK-3, CCL14 / HCC-1 / HCC-3, CCR1, EN-RAGE, eotaxina-3 / CCL26, FGF R4, FGF-10 / KGF-2, FGF-19, FGF-21, Flt-3 Ligando, folistatina 1, GASP-1 / WFIKKNRP, GCP-2 / CXCL6, GFR alfa-3, GREMLIN, GRO-a, HGF, HRG-beta 1, I-309, ICAM-1, IFN-alfa / R2 beta, IGFBP-2, IGF-I, IL-4, IL-5 R alfa, IL-10 R beta, IL-12 R beta 1, IL-13 R alfa 2, IL-20 R eta, IL-22 BP, IL-23 R, FACX, LIF, LIF R alfa, LIGHT / TNFSF14, lipocalina-1, lipocalina-2, LRP-1, MCP-4 / CCL13, M-CSF, MDC, MFG-E8, MICA, MIP-1b, MIP-1d, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-12, MMP-16 / MT3-MMP, MMP-25 / MT6-MMP, NAP-2, NeuroD1, PDGF-AB, PDGF-BB, PDGF-C, PDGF-D, pentraxina 3 / TSG-14, persefina, PF4 / CXCL4, PLUNC, P-selectina, RANTES, RELM beta, ROBO4, S100A10, SAA, SCF, SIGIRR, Smad 1, Smad 5, Smad 8, Prdx6, Tarc, TCCR / WSX-1, TGF-beta 3, TGF-beta 5, Tie-2, TIMP-1, TROY / TNFRSF19, uPA - 1, 75-fold or more: angiopoietin-1, angiopoietin-4, BIK, CCR1, FGF-21, GRO-a, HGF, IL-10 R beta, IL-12 R beta 1, MCP-4 / CCL13, MIP-1b, MIP-1d, NeuroD1, PDGF-C, Prdx6, TIMP-1, uPA - 2-fold or more: BIK, GRO-a, HGF, MCP-4 / CCL13, uPA In conclusion, the inventors of the present description have prepared neuronal regeneration-promoting cells from mesenchymal stem cells derived from tonsils and adipose tissue and have investigated their expression pattern using a CD marker assay. Furthermore, they have identified the neuronal regeneration effect of these neuronal regeneration-promoting cells. This suggests that tonsil tissue, which has been discarded as medical waste, can be used to prepare cells that have a neuronal regeneration effect. The neuronal regeneration-promoting cells described herein can be used in various ways in the field of neuronal regeneration. Although the specific illustrative embodiments of the present description have been described, those of ordinary skill in the art may modify and change the present description in various ways by adding, changing, deleting, etc. without departing from the scope of the present description as defined by the appended claims.

Claims

1. A method for screening neuronal regeneration-promoting cells with neuronal regeneration activity from differentiated mesenchymal stem cell cells, comprising: a cell screening step in which one or more markers selected from a group consisting of CD121a, CD106, and CD112 are upregulated among the differentiated cells compared to pre-differentiated mesenchymal stem cells.

2. A method for screening neuronal regeneration-promoting cells with neuronal regeneration activity from differentiated mesenchymal stem cell cells, comprising: a cell screening step in which one or more markers selected from a group consisting of CD26 and CD141 are downregulated among the differentiated cells compared to pre-differentiated mesenchymal stem cells. 3.The method for screening neuronal regeneration-promoting cells according to claim 1 or 2, wherein the mesenchymal stem cells are derived from tonsils or fat.

4. The method for screening neuronal regeneration-promoting cells according to claim 1 or 2, wherein the differentiated cells are differentiated from neurospheres formed by culturing mesenchymal stem cells.

5. The method for screening neuronal regeneration-promoting cells according to claim 1 or 2, wherein the neuronal regeneration activity comprises the myelination of peripheral nerves. 6.Neuronal regeneration-promoting cells screened by the screening method according to claim 1 or 2, differentiated from tonsil-derived mesenchymal stem cells, the neuronal regeneration-promoting cells having the following characteristics: a) the expression of the markers CD121a, CD106, and CD112 is upregulated compared to tonsil-derived mesenchymal stem cells; and b) the expression of the markers CD26 and CD141 is downregulated compared to tonsil-derived mesenchymal stem cells.

7. The neuronal regeneration-promoting cells according to claim 6, wherein, in the neuronal regeneration-promoting cells, the expression of the marker CD121a is upregulated by 30% or more compared to tonsil-derived mesenchymal stem cells. 8.

1. Neuron-promoting cells according to claim 6, wherein, in the neuronal-promoting cells, the expression of the marker CD106 is upregulated by 5% or more compared to tonsil-derived mesenchymal stem cells.

9. Neuron-promoting cells according to claim 6, wherein, in the neuronal-promoting cells, the expression of the marker CD112 is upregulated by 10% or more compared to tonsil-derived mesenchymal stem cells.

10. Neuron-promoting cells according to claim 6, wherein, in the neuronal-promoting cells, the expression of the marker CD26 is downregulated by 5% or more compared to tonsil-derived mesenchymal stem cells. 11.The neuronal regeneration-promoting cells according to claim 6, wherein, in the neuronal regeneration-promoting cells, the expression of the marker CD141 is downregulated by 5% or more compared to tonsil-derived mesenchymal stem cells.

12. A pharmaceutical composition for preventing or treating a neurological disease, comprising the neuronal regeneration-promoting cells according to claim 6 as an active ingredient, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, wherein the neurological disease is one or more diseases selected from a group consisting of Charcot-Marie-Tooth neuropathy, diabetic peripheral neuropathy, spinal cord injury, amyotrophic lateral sclerosis, carpal tunnel syndrome, infantile paralysis, leprosy, muscular dystrophy, polymyositis, and myasthenia gravis. 14.The pharmaceutical composition according to claim 12, wherein the neuronal regeneration activity comprises the myelination of peripheral nerves.

15. The neuronal regeneration-promoting cells according to claim 6 for use in a method of treating a neurological disease.

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