Composition for prevention or treatment of neurological disease comprising: schwann cell precursor (SCP) or schwann cell (SC) differentiated therefrom; and natural killer (NK) cells

Abstract

The present invention relates to a pharmaceutical composition and a cellular therapeutic agent for the prevention or treatment of a neurological disease, comprising as active ingredients: a Schwann cell precursor (SCP) prepared from a pluripotent stem cell (PSC) or a somatic cell, or a Schwann cell (SC) differentiated therefrom; and natural killer (NK) cells. Specifically, the SCP expresses any one or more selected from the group consisting of GAP43, SOX10, IGFBP2, and a combination thereof, the SC expresses any one or more selected from the group consisting of S100B, SOX10, and a combination thereof, and the NK cells express any one or more selected from the group consisting of CD56+, CD16+, and a combination thereof.

Description

A composition for preventing or treating a neurological disease, comprising Schwann cell precursor (SCP) or Schwann cell (SC) differentiated therefrom; and natural killer cells (NK) cells.

[0001] The present invention relates to a pharmaceutical composition and a cell therapy agent for preventing or treating a neurological disease, which contain as active ingredients pluripotent stem cells (PSCs) or Schwann cell precursors (SCPs) prepared from somatic cells or Schwann cells (SCs) differentiated therefrom; and natural killer (NK) cells. Specifically, the Schwann precursor cells (SCPs) express at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and a combination thereof, the Schwann cells (SCs) express at least one selected from the group consisting of S100B, SOX10, and a combination thereof, and the natural killer (NK) cells express CD56. + , CD16 + and characterized by expressing at least one selected from the group consisting of combinations thereof.

[0002]

[0003] Schwann cells (SCs) are essential glial cells in the peripheral nervous system (PNS) that play a pivotal role in supporting neurons and promoting nerve repair. Schwann cells are responsible for forming myelin, which insulates nerve fibers in the PNS and ensures rapid transmission of nerve impulses. Schwann cell-mediated myelination is crucial not only for normal nerve function but also for the repair and regeneration of damaged nerves. In addition to their role in myelination, Schwann cells secrete various neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3), which promote nerve survival, enhance axon growth, and support nerve regeneration after injury. Schwann cells also create components of the extracellular matrix that provide support to promote axon guidance and regrowth, creating an environment conducive to nerve repair.

[0004] Due to the unique properties of human Schwann cells, they are very useful for disease modeling, drug discovery, and the development of new treatments such as cell therapy. However, securing sufficient cells that maintain functionality is difficult. For primary cultured human Schwann cells, the isolation efficiency and culture purity vary depending on the condition of the nerve biopsy, and collection requires an invasive procedure that carries risks such as pain and potential nerve damage. The low proliferative capacity of Schwann cells in ex vivo culture makes it difficult to expand to a sufficient number of cells. In addition, the purity of Schwann cells may decrease over time due to increased contamination with fibroblasts, which may deteriorate their quality. Although it is important for cultured Schwann cells to maintain their original characteristics (such as myelination capacity and neurotrophic factor secretion), long-term culture and expansion in vitro can lead to a loss of Schwann cell characteristics and function.

[0005] Alternatively, active research and development is underway to optimize and expand the production of Schwann cells from stem cells. In particular, human pluripotent stem cells (PSCs), including human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs), are attracting attention as important resources for the differentiation and production of Schwann cells due to their excellent proliferative and differentiation capacities. During development, Schwann cells exist in the following forms: 1) neural crest (stem) cells (NC(S)Cs), 2) Schwann cell precursor cells (SCPs), 2) immature, unmyelinated Schwann cells, and 3) mature, myelinated Schwann cells. Typically, Schwann cells are obtained from PSCs by first differentiating multipotent neural crest stem cells (NCSCs), the developmental precursors of Schwann cells, and then redifferentiating NCSCs into Schwann cells. However, differentiating PSCs into Schwann cells via NCSCs has the following limitations: 1) the differentiation process is complex and time-consuming, 2) productivity and purity are low, and 3) biological function and therapeutic efficacy are low.

[0006] Specifically, Schwann progenitor cells (SCPs) are an intermediate cell type that exists between neural crest cells that appear in the early stages of development and the pre-myelin Schwann cell stage. Schwann progenitor cells (SCPs) obtained from PSCs are capable of proliferation and culture, and their importance as an optimal Schwann cell source that can directly produce Schwann cells in a short period of time is highlighted. Therefore, this technology produced Schwann progenitor cells (PSC-SCPs) and Schwann cells (PSC-SCP-SCs) from PSCs and analyzed their effects in treating nerve damage / diseases.

[0007] Meanwhile, natural killer (NK) cells are a type of lymphocyte blood cell that plays an important role in innate and adaptive immune responses. In particular, they have the function of recognizing and immediately eliminating abnormal cells that cause diseases, such as cancer cells, viruses, bacteria, fungi, and parasites, by detecting abnormal proteins on the cell surface or a decrease in major histocompatibility complex (MHC) I molecules without the need to recognize specific antigens, making them an important target for the development of therapeutics for various diseases.

[0008] NK cells can promote nerve recovery / regeneration by removing damaged nerves, suppress neuroinflammation and autoimmune diseases by influencing other immune cells such as microglia and T cells, and suppress the spread of infection within nerve tissue by eliminating nerve cells infected with viruses. Therefore, the potential usefulness of NK cells in the treatment of various neurological diseases, such as nerve damage diseases and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, is being highlighted. However, research and technological development on their direct role and potential therapeutic effects are insufficient.

[0009] Recently, with the advancement of direct reprogramming technology, efforts are actively underway to directly produce functional cells with high clinical utility, without going through stem cell production processes such as prostate cancer stem cells (PSCs). Functional cells produced through direct reprogramming technology have significant technological advantages, including a lower risk of epigenetic remodeling and tumorigenesis, and the simplified cell production process facilitates increased safety, reliability, and efficiency. These characteristics are expected to dramatically reduce the time and cost required for therapeutic development, thereby contributing to the reduction of barriers to commercialization. Consequently, R&D efforts to secure raw materials for cell therapy for various diseases are continuously increasing. Therefore, this technology was used to analyze the efficacy of Schwann progenitor (drSCP) cells and NK (drNK) cells obtained through direct reprogramming in the treatment of nerve damage and diseases.

[0010] To date, there has been no report on the preventive, therapeutic, or ameliorating effects on neurological diseases when combining Schwann progenitor cells (PSC-SCP) or Schwann cells differentiated therefrom (PSC-SCP-SC), or somatic cell reprogramming-induced Schwann progenitor cells (drSCP) or Schwann cells differentiated therefrom (drSCP-SC), and NK cells.

[0011]

[0012] The present inventors have endeavored to develop a method for producing a treatment for nerve damage / disease with rapid, high production efficiency, and improved function. As a result, they have produced Schwann progenitor cells capable of in vitro proliferation through differentiation culture from human pluripotent stem cells or direct reprogramming culture from somatic cells, and confirmed that when differentiation of the Schwann progenitor cells into Schwann cells is induced, human Schwann cells with improved functionality in vivo and in vitro can be produced in a shortened time and under conditions with improved production efficiency. In addition, they have confirmed that this can be usefully used for the prevention or treatment of nerve diseases when combined with human natural killer (NK) cells, thereby completing the present invention.

[0013]

[0014] One object of the present invention is to provide a Schwann progenitor cell (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and a combination thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10, and a combination thereof; and CD56. + , CD16 + The present invention provides a pharmaceutical composition for preventing or treating a neurological disease, comprising natural killer (NK) cells expressing at least one selected from the group consisting of: and combinations thereof.

[0015] Another object of the present invention is to provide a Schwann progenitor cell (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and a combination thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10, and a combination thereof; and CD56. + , CD16 +The present invention provides a cell therapy composition for preventing or treating a neurological disease, comprising natural killer (NK) cells expressing at least one selected from the group consisting of: and combinations thereof.

[0016] Another object of the present invention is to provide a Schwann progenitor cell (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and a combination thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10, and a combination thereof; and CD56. + , CD16 + The present invention provides a method for preventing or treating a neurological disease, comprising administering to a subject other than a human a pharmaceutical composition for preventing or treating a neurological disease, the pharmaceutical composition comprising natural killer (NK) cells expressing at least one selected from the group consisting of: and combinations thereof, to a subject suspected of having a neurological disease.

[0017]

[0018] Schwann progenitor cells (SCPs) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and a combination thereof, or Schwann cells (SCs) expressing at least one selected from the group consisting of S100B, SOX10, and a combination thereof; and CD56 + , CD16 + When natural killer (NK) cells expressing at least one selected from the group consisting of and combinations thereof were treated alone or in combination in a peripheral and central nervous system injury disease model, it was confirmed that they had different gene expression characteristics from existing Schwann progenitor cells (NCSCs), had superior neurotrophic factor GDNF secretion ability compared to existing NCSCs, and had excellent regenerative and therapeutic effects on the damaged nervous system. Therefore, they can be usefully used in the prevention or treatment of neurological diseases.

[0019]

[0020] Figure 1 is a schematic diagram of the differentiation induction process from human pluripotent stem cells (hPSCs) to Schwann progenitor cells (hSCPs), and shows the development results for Protocols 4 and 5, which are improved by introducing new factors compared to the existing control Protocol 1. Figure 1A is a schematic diagram showing the differentiation of human pluripotent stem cells (hPSCs) to Schwann progenitor cells (hSCPs) of the existing control (Protocol 1) and the present invention (Protocol 2-8). Figure 1B is a diagram showing different differentiation medium compositions used in Stages 1 and 2 of Figure 1A. Figure 1C is the result of quantitative analysis of the expression level of the SOX10 gene from total RNA of cells on day 24 according to the differentiation induction method of Figure 1B. Figure 1D shows the results of quantitative analysis of the gene expression levels of CD49d, ERBB3, and PLP1 in the groups in which SOX10 was confirmed to have increased in protocols 1, 4, 5, and 7 in Figure 1C. Figure 1E shows the results of immunocytochemistry to confirm the protein expression of SOX10, GAP43, and IGFBP2 in SCP differentiated by the existing (protocol 1) and the new method by introducing new factors (Protocols 4 and 5).

[0021] Figure 2 shows the results of quantitative analysis of gene expression of Schwann cell markers S100b, NGFR, MPZ, and EGR2 in Schwann cells (Protocol 1-SC, Protocol 4-SC) differentiated from Schwann precursor cells produced using Protocol 1 and 4 of Figure 1 and Schwann cells (iSCP-SC) produced through differentiation-induced culture from SCP (iSCP) produced by somatic cell reprogramming technique (Figure 2A), and Figure 2B shows the results of confirming SOX10 and S100B protein expression in the differentiated Schwann cells of Figure 2A.

[0022] FIG. 3 shows the results confirming that the expression of neurotrophic factors GDNF and IGFBP-2 is higher in the induced SCP of the present invention compared to the existing Schwann precursor cells (NCSC). FIG. 3A shows the results of quantitative analysis of the gene expression levels of GDNF and IGFBP-2 in H9 (hPSC), NCSC, and SCP, respectively, and FIG. 3B shows the results of quantitative analysis of the protein secretion levels of GDNF and IGFBP-2 in the conditioned medium (CM) of NCSC and SCP, respectively.

[0023] Figure 4 shows the cell phenotypic characteristics of the induced natural killer (drNK) cells of the present invention, and Figure 4A shows four representative types of NK cells showing different CD56 and CD16 expression patterns to compare and analyze the effect of the damaged nerve target natural killer cells of the present invention: 1. The main cell type of PBMC-derived NK cells CD56 dim pNK, 2. IL-2 / IL-15 cytokine activation CD56 bright pNK, 3. immortalized NK cell line NK92, 4. drNK list produced through direct reprogramming of the present invention, Fig. 4B shows the results of flow cytometry analysis of CD56 and CD16 expression patterns of each NK cell, confirming that the four types of NK cells show different characteristics. Fig. 4C shows CD56 dim The results of comparing the expression of cell surface receptors related to NK cytotoxicity in pNK and drNK cells using flow cytometry.

[0024] Figure 5 shows the results of quantitative analysis of the gene expression levels of cytokines expressed in drNK using qRT-PCR, and confirms that 10 cytokines (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) are highly expressed in drNK compared to the control group NK-92 and iPS-NK.

[0025] Figure 6 shows each CD56 using a human proteome cytokine array. dim As a result of identification from the conditioned medium of pNK and drNK cells, Figure 6A shows the results of quantitative analysis of 56 types of secreted proteins in the conditioned medium of drNK cells, and Figure 6B shows CD56. dim As a result of quantitative analysis of 28 types of increased secreted proteins in the conditioned medium of drNK cells compared to the conditioned medium of pNK cells, Figure 6C shows the results of confirming DPP4, M-CSF, and BDNF, which are proteins specifically secreted in the conditioned medium of drNK cells.

[0026] Figure 7 shows the results of confirming the clearing effect of drNK and verifying the dependence of CD16 expression. Figure 7A shows the SH-SY5Y neuronal cell model with ROS positive labeling (MitoSox Red) due to H2O2-mediated damage, Figure 7B shows the results of confirming the significantly superior clearing effect of drNK compared to the control group under co-culture conditions with NK cells and ROS positive damaged neurons, Figure 7C shows a schematic diagram of a cytotoxicity assay using an anti-CD16 antibody, and Figure 7D shows the results of analyzing the correlation between the clearing effect and CD16 expression, confirming that the clearing effect was most affected by the CD16 antibody in drNK cells with the highest expression of CD16. Therefore, the clearing activity of drNK cells is confirmed to be associated with CD16 expression.

[0027] Figure 8 shows the results confirming the significantly superior effect on neurite outgrowth by SCP / SCP-SC and drNK cells compared to the control group. Figure 8A is a schematic diagram of the damaged nerve recovery / regeneration assay through treatment with NGF as a control substance for nerve regeneration in axotomy or partial nerve injury model, SCP, SC, NCSC, and NK cells alone or in co-culture. Figure 8B is the results confirming the neurite length recovery / regeneration effect by drNK, SCP-SC, and drNK+SCP-SC alone or in co-culture after neurite cut injury in stem cell-derived nerve cells through cell phase contrast microscopic images and TUJ1 positive staining images, showing not only the nerve regeneration promotion effect by drNK and SCP-SC but also the synergistic effect of nerve regeneration by drNK+SCP-SC. Figure 8C is the results showing the synergistic effect of drNK+SCP-SC on nerve regeneration as a control in a partial nerve injury model with NGF, CD56 dim The results of comparative analysis of the neurite damage recovery / regeneration effect by single and combined treatment of SCP-differentiated SC (SCP-SC) and drNK of the present invention compared to pNK and primary cultured Schwann cells (pSC) are shown in Figure 8D. The results are compared between the conventional control SCP (NCSC) and the conventional control CD56 of the present invention and SCP of the present invention. dim This is the result of a comparative analysis of the neurite damage recovery / regeneration effects of pNK and drNK of the present invention.

[0028] FIG. 9 shows the results of confirming the promotion and treatment effect of sciatic nerve regeneration by SCP and NK cell single or combined transplantation in an animal model, and FIG. 9A is a schematic diagram of an animal model experiment, and FIGS. 9B and 9C show the superior GFP+ SCP influx / engraftment effect of SCP and the increase in myelination marker MBP-positive myelinated cells compared to the control group NCSC, and FIG. 9D shows the results of analyzing the recovery of motor function according to nerve regeneration in the animal model of FIG. 10A through a Rotarod test, confirming that the motor ability was significantly improved in the group transplanted with the SCP of the present invention compared to the control group NCSC, and FIG. 9E shows the results of showing the significantly superior nerve regeneration effect by combined treatment of SCP+drNK compared to the single treatment.

[0029] Figure 10 shows the results showing the promotion of nerve regeneration and therapeutic effects by SCP-SC and NK cell transplantation alone or in combination in a sciatic nerve partial injury animal model. Figure 10A shows the results showing the effects of NGF, CD56 in a sciatic nerve partial injury model. bright Four weeks after single and combined transplantation of pNK, drNK, and SCP-SC cells, the sciatic nerves of each group were sampled and stained with hematoxylin and eosin (H&E) to confirm the therapeutic effect on peripheral nerve diseases. In the single treatment group, the existing CD56 bright As a result of confirming that the drNK of the present invention has a superior nerve recovery / regeneration effect compared to NK, and at the same time, it can be confirmed that the SCP-SC+NK combination treatment group has a relatively better regenerative treatment effect compared to the single treatment group, the treatment effect after the existing control drug NGF treatment, NK, SCP-SC alone, and combination transplantation was confirmed by quantitative analysis of the immunohistochemical staining positive images of the nerve cell marker TUJ1 as a result of confirming the treatment effect after the existing control drug NGF treatment, NK, SCP-SC, CD56 compared to the existing control drug NGF bright pNK and drNK single treatment groups showed significant nerve regeneration promotion effects and at the same time, existing CD56 brightThis is a result confirming that the nerve recovery / regeneration effect of drNK of the present invention is superior to that of pNK.

[0030] Figure 11 is a diagram showing the results of promoting nerve regeneration and therapeutic effects by single or combined transplantation of SCP-SC and NK cells in the animal model of Figure 10. Figure 11A shows the results of immunostaining for Schwann cell marker S100 in nerve bundles at the injured site, Figure 11B shows the results of quantitative analysis of the expression of the S100 gene in total mRNA obtained from nerve bundles at the injured site, Figure 11C shows the results of immunostaining for myelin marker MBP in nerve bundles at the injured site, and Figure 11D shows the results of quantitative analysis of the expression of the MBP gene in total mRNA obtained from nerve bundles at the injured site. As a result of performing myelin marker MBP immunohistochemical staining and qRT-PCR analysis, it was confirmed that S100 and MBP positive cells increased most significantly in the SCP-SC+drNK composite transplantation group compared to the SCP-SC and drNK cell-only transplantation group. This result confirms that myelin regeneration was most enhanced in the SCP-SC and drNK composite transplantation group, which contributed to the improvement of nerve function.

[0031] Figure 12 shows the results of a behavioral experiment (Rotarod) in the animal model of Figure 10 showing the effects of promoting nerve regeneration and treatment by SCP-SC and NK cell transplantation alone or in combination. The SCP-SC transplantation group showed the best motor function improvement effect among the single transplantation groups, and CD56 bright The results confirmed that the drNK transplant group had a better effect on improving motor function compared to the pNK group, and that the drNK+SCP-SC composite transplant group showed the best improvement in motor function among the comparison groups.

[0032]

[0033] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0034]

[0035] One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating a neurological disease, comprising Schwann precursor cells (SCPs) or Schwann cells (SCs) differentiated therefrom; and natural killer (NK) cells as active ingredients.

[0036] Specifically, Schwann progenitor cells (SCPs) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof, or Schwann cells (SCs) expressing at least one selected from the group consisting of S100B, SOX10, and combinations thereof; and CD56. + , CD16 + A pharmaceutical composition for preventing or treating a neurological disease is provided, comprising natural killer (NK) cells expressing at least one selected from the group consisting of: and combinations thereof.

[0037] The present inventors have first discovered that when the composition is administered to a peripheral and central nervous system damage disease model, it has different gene expression characteristics from existing Schwann progenitor cells (NCSCs), has superior neurotrophic factor GDNF secretion ability compared to existing NCSCs, and has excellent regenerative and therapeutic effects on the damaged nervous system.

[0038] In particular, the Schwann precursor cells (SCP) of the present invention were found to have significantly higher expression levels of neurotrophic factor GDNF and IGFBP-2 genes compared to NCSC, a representative Schwann precursor cell known to the public, and to have higher secretion levels of proteins that affect nerve regeneration and growth.

[0039] In addition, the induced natural killer (drNK) cells of the present invention have higher expression of NK cell activating receptors such as CD69, NKG2D, DNAM-1, and NKp46 compared to the control NK, and 10 types of cytokine / chemokine genes overexpressed in the drNK of the present invention were confirmed, and 3 types of proteins specifically identified only in drNK were confirmed.

[0040] Furthermore, when the Schwann precursor cells (SCP) of the present invention or Schwann cells (SC) differentiated therefrom and natural killer (NK) cells were combined and treated in a peripheral and central nervous system injury disease model, it was confirmed that the SCP-SC and NK combination treatment group had superior nerve growth and regenerative treatment effects compared to the single treatment group.

[0041] In particular, it was confirmed that the induced natural killer (drNK) cells of the present invention, when treated with peripheral and central nervous system damage disease models compared to the control NK, had relatively superior nerve growth and regenerative treatment effects in the SCP-SC and NK complex treatment group compared to the single treatment group.

[0042]

[0043] This suggests that the composition of the present invention has a higher level of gene expression or protein secretion affecting nerve regeneration or growth compared to the previously known SCP or NK cells, and is superior in nerve growth and regeneration effect when combined with these compared to their single treatment, suggesting that it is useful for preventing or treating nerve diseases.

[0044]

[0045] As a specific example, the Schwann progenitor cell (SCP) of the present invention may be produced by a method for producing SCP from PSC, which comprises the steps of (a) culturing pluripotent stem cells in a first medium containing SB431542 and CT99021; and (b) culturing the cells cultured in step (a) in a second medium to which NRG1 (Neuregulin-1) is additionally added, but is not limited thereto.

[0046] The term "SB431542" of the present invention is a specific inhibitor of TGF-β (Transforming growth factor-β) and has a structure represented by the following chemical formula 1.

[0047] [Chemical Formula 1]

[0048]

[0049] Specifically, the SB431542 may be included at a concentration of, but not limited to, 1 to 100 μM, more specifically 1 to 50 μM, more specifically 1 to 30 μM, and even more specifically 5 to 25 μM.

[0050] The term "CT99021" of the present invention refers to CHIR-99021 (CT99021), which is a GSK-3α / β inhibitor and may also be named CT99021, CHIR99021, CHIR 99021, CHIR-99021 or CT-99021. It has a structure represented by the following chemical formula 2.

[0051] [Chemical Formula 2]

[0052]

[0053]

[0054] Specifically, the CT99021 may be included at a concentration of 1 to 100 μM, more specifically 1 to 50 μM, more specifically 1 to 10 μM, and even more specifically 1 to 5 μM, but is not limited thereto.

[0055] The "NRG1 (Neuregulin-1)" of the present invention is a protein encoded by the NRG1 gene and acts on the EGFR receptor. Specifically, the NRG1 may be included at a concentration of 1 to 1000 ng / ml, more specifically 10 to 500 ng / ml, more specifically 20 to 200 ng / ml, and even more specifically 30 to 100 ng / ml, but is not limited thereto.

[0056]

[0057] In another specific example, the first medium of step (a) may additionally include FGF2, and the second medium of step (b) may additionally include StemRegenin I (SR I), but is not limited thereto.

[0058] The "FGF2 (Fibroblast growth factor 2)" of the present invention may be used interchangeably with the terms bFGF (basic fibroblast growth factor) or FGF-β as a fibroblast growth factor. Specifically, the FGF2 may be included at a concentration of 1 to 100 μg / ml, more specifically 1 to 50 μg / ml, more specifically 5 to 50 μg / ml, and even more specifically 10 to 30 μg / ml, but is not limited thereto.

[0059] The "StemRegenin I (SR I)" of the present invention is an aryl hydrocarbon receptor inhibitor, and refers to (4-(2-(2-(Benzo[b]thiphen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol hydrochloride). The StemRegenin I may be additionally included in the second medium for producing Schwann progenitor cells (SCPs). The SR I may be included at a concentration of 1 to 100 μM, specifically 1 to 50 μM, more specifically 1 to 10 μM, and even more specifically 1 to 5 μM, but is not limited thereto.

[0060] Another specific aspect of the present invention provides Schwann progenitor cells (SCPs) produced by the above method. Schwann progenitor cells produced by the method of the present invention are Schwann progenitor cells differentiated from pluripotent stem cells (PSCs), and the Schwann progenitor cells have multipotency capable of differentiating into Schwann cells, melanocytes, etc., a high proliferation rate (expandability), and the potential for long-term maintenance. In addition, the Schwann progenitor cells may express at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof, which are Schwann progenitor cell-specific marker genes, but are not limited thereto.

[0061] The term "Schwann cell precursor (SCP)" of the present invention refers to an intermediate stage that Schwann cells pass through during neural crest development, specifically, an intermediate stage cell between neural crest (stem) cells (NC(S)C) and immature pre-myelin Schwann cells. The Schwann precursor cells can differentiate into Schwann cells.

[0062] The term "pluripotent stem cell (PSC)" of the present invention refers to an undifferentiated stem cell that has the ability to differentiate into all cells of the three germ layers (endoderm, mesoderm, and ectoderm). Under in vitro culture conditions, undifferentiated pluripotent stem cells have pluripotency and self-renewal ability while maintaining a normal karyotype. In the present invention, pluripotency may include both pluripotency and multipotency. Pluripotent stem cells may include embryonic carcinoma (EC) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, etc. Specifically, the pluripotent stem cell of the present invention may be a human-derived ES cell (hESC) or an induced pluripotent stem (iPS) cell (hiPSC), but any species of origin is included without limitation as long as it has pluripotency.

[0063] In one specific example of the present invention, the difference between the SCP of the present invention and neural crest stem cells (NCSCs), which are representative Schwann precursor cells known in the art, was confirmed, and the expression levels of neurotrophic factors GDNF and IGFBP-2 genes were confirmed to be significantly higher in SCPs than in NCSCs, and the secretion level of proteins affecting nerve regeneration and growth was confirmed to be high. Through this, it can be seen that the nerve growth factor secreted in high levels in the SCP of the present invention compared to the existing NCSCs can exhibit a greater effect on nerve growth and regeneration.

[0064]

[0065] In another specific example, the human pluripotent stem cell-Schwann precursor cell-derived Schwann cell (PSC-SCP-SC) of the present invention may be produced by a method for producing Schwann cells from PSC, comprising the steps of: (a) culturing pluripotent stem cells in a first medium containing SB431542 and CT99021; (b) culturing the cells cultured in step (a) in a second medium to which NRG1 (Neuregulin-1) is additionally added; (c) recovering SCP from the cultured medium; and (d) culturing the recovered SCP in a third medium containing FBS and NRG1.

[0066] In another specific example, the third medium of step (d) may additionally include, but is not limited to, one or more selected from the group consisting of retinoic acid, forskolin, and PDGF-BB.

[0067] The term "retinoic acid" of the present invention is a metabolic product produced when vitamin A is broken down in the body, and C 20 H 28 It has the chemical formula of O2. It is known to have effects such as suppressing colon cancer and treating rheumatism. Specifically, the retinoic acid may be included in the medium at a concentration of 1 to 300 nM, more specifically, 10 to 200 nM, and more specifically, 50 to 150 nM, but is not limited thereto.

[0068] The term "Forskolin" of the present invention is a labdane diterpene produced from the Indian Coleus plant (Plectranthus barbatus). Specifically, the forskolin may be included in the medium at a concentration of 1 to 100 μM, more specifically 1 to 50 μM, and even more specifically 1 to 10 μM, but is not limited thereto.

[0069] The term "PDGF-BB (Platelet-derived growth factor-BB)" of the present invention refers to a dimer of PDGFB encoded by the PDGFB gene. Specifically, the PDGF-BB may be included in the medium at a concentration of 1 to 100 ng / ml, more specifically, 1 to 50 ng / ml, and even more specifically, 5 to 15 ng / ml, but is not limited thereto.

[0070] The term "Schwann cell (SC)" of the present invention is a glial cell in the peripheral nervous system, which plays a role in myelin formation, nerve impulse transmission, and neurotrophic factor secretion, and is known to affect the survival of nerves and the growth of axons in particular.

[0071] Another aspect of the present invention provides Schwann cells produced by the above method. Schwann cells produced by the method of the present invention are Schwann cells produced from pluripotent stem cells via Schwann progenitor cells, and exhibit positive expression of Schwann cell-specific marker genes such as S100B and SOX10.

[0072] In another specific example, the natural killer (NK) cells are CD56 + , CD16 + and combinations thereof. Specifically, the natural killer (NK) cells may express at least one selected from the group consisting of CD56. dim , CD56 bright, CD56superbright, CD16 dim , CD16 bright , CD16superbright, and combinations thereof, but is not limited thereto.

[0073] For example, (1) pNK isolated from human peripheral blood, (2) CD56 isolated from human peripheral blood dim CD16 bright pNK, (2) CD56 activated by IL-2 / IL-15 cytokines of pNK isolated from human peripheral blood bright CD16 bright pNK, (3). Immortalized CD56 bright CD16 dim It may be, but is not limited to, NK cell lines (NK92).

[0074] In another specific example, the directly reprogrammed NK (drNK) cells may be produced by a method for producing induced natural killer cells from isolated cells, comprising: (a) introducing a reprogramming factor into isolated cells; (b) culturing the cells of step (a) in a first medium containing i) cytokines, growth factors, and GSK3β (Glycogen synthase kinase 3β) inhibitors to increase the efficiency of direct reprogramming, starting from the day after introducing the reprogramming factors; and (ii) culturing the cells in a second medium containing cytokines, growth factors, and AHR (Aryl hydrocarbon receptor) inhibitors to promote the production of induced natural killer (drNK) cells.

[0075] Another specific aspect of the present invention provides induced natural killer cells (drNK) produced by the above method. The induced natural killer cells produced by the method of the present invention may express at least one selected from the group consisting of CD56superbright, CD16superbright, and combinations thereof, but are not limited thereto.

[0076] In another specific example, the induced natural killer (drNK) cells may overexpress any one or more genes selected from the group consisting of CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, and LTB compared to a control, but are not limited thereto.

[0077] In another specific example, the induced natural killer (drNK) cells may express one or more proteins selected from the group consisting of, but not limited to, DDP4, M-CSF, and BDNF.

[0078] The term "cytokine," as used herein, refers to a variety of relatively small proteins produced by cells and used in cell signaling, which can affect other cells, including the cell itself. It is generally associated with, but not limited to, immune responses to inflammation or infection. Specifically, the cytokines may include, but are not limited to, IL-2, IL-3, IL-5, IL-6, IL-7, IL-11, IL-15, BMP4, Acivin A, Notch ligand, G-CSF, and SDF-1.

[0079] In the present invention, the term "growth factor" means a polypeptide that promotes division, growth, and differentiation of various cells, and includes, but is not limited to, epidermal growth factor (EGF), platelet-derived growth factor-AA (PDGF-AA), insulin-like growth factor-1 (IGF-1), transforming growth factor-β (TGF-β), or fibroblast growth factor (FGF).

[0080] For the purposes of the present invention, cytokines and growth factors are included in a medium that directly reprograms isolated cells into lineage-converted cells, and are not limited to the types of cytokines and growth factors as long as they are used for direct reprogramming.

[0081] The term "natural killer (NK) cell" of the present invention is a key innate immune cell that immediately recognizes and eliminates infections by viruses, bacteria, fungi, and parasites, as well as abnormal self-cells. Unlike T cells that recognize target cells by expressing antigen-specific receptors, NK cells recognize abnormal changes in target cells, such as the balance of inhibitory or activating receptors such as killer immunoglobulin receptors (KIR), natural cytotoxicity receptors (NCR), DNAM-1 (DNAX accessory molecule-1), and NKG2D (NK group 2 member D), loss of surface MHC (Major histocompatibility complex) class I antigens, and accumulation of abnormal proteins, without specificity for antigens and human leukocyte antigen (HLA) matching, and exhibit contact-dependent cytotoxicity through various mechanisms. Unlike T cells, which can cause graft-versus-host disease (GVHD) against non-self allogeneic cells with mismatched human leukocyte antigen (HLA), allogeneic NK cells have been shown to have little side effects of graft-versus-host disease and rather a strong therapeutic effect.

[0082] In the present invention, the term "direct reprogramming" refers to a method of converting a lineage into a desired cell with completely different characteristics by controlling the global gene expression pattern of a specific cell. The direct reprogramming may include, but is not limited to, cell reprogramming, differentiation, direct differentiation, dedifferentiation, direct dedifferentiation, conversion, direct conversion, trans-differentiation, or direct trans-differentiation.

[0083] The above direct reprogramming may be a "cell transformation" performed by introducing an oligonucleotide or vector containing a foreign gene or DNA into a cell, and may mean that the cell changes into a different state. The above "differentiation" refers to a phenomenon in which daughter cells produced by cell division acquire functions different from those of the original parent cell, and in the present invention, the above "direct reprogramming" may be used interchangeably with "direct cell transformation induction", "direct cell transformation", and "cell transformation".

[0084] For the purposes of the present invention, natural killer cells are those obtained through direct reprogramming, and may be used interchangeably with directly reprogrammed natural killer (drNK) cells.

[0085] The term "isolated cell" of the present invention has no particular limitations, but may be a cell whose lineage has already been specified, such as a germ cell, a somatic cell, or a progenitor cell. The "somatic cell" refers to all cells that have completed differentiation that constitute animals and plants, excluding germ cells, and the "progenitor cell" refers to a parent cell that does not express a differentiation trait but has that differentiation fate, when the cell corresponding to the offspring is found to express a specific differentiation trait. For example, for blood cells, hematopoietic stem cells correspond to progenitor cells, and for mesenchymal cells, mesenchymal stem cells correspond to progenitor cells.

[0086] The isolated cells may be derived from humans, but are not limited thereto, and cells derived from various organisms may also fall within the scope of the present invention. Furthermore, the isolated cells of the present invention may include both in vivo and ex vivo cells.

[0087] For example, the isolated cell may be a somatic cell, or as another example, a somatic cell other than an NK cell, or as another example, at least one selected from the group consisting of a blood cell and a fibroblast, but is not limited thereto. For example, the blood cell may be a peripheral blood mononuclear cell (PBMC), but is not limited thereto.

[0088] In the present invention, the term "direct reprogramming induction factor" refers to a gene (or polynucleotide) that can induce cell transformation when introduced into a cell, or a protein encoded therefrom. The direct reprogramming induction factor may vary depending on the target cell to be obtained through reprogramming and the type of cell before cell transformation. Cell transformation using the direct reprogramming induction factor induces transformation into a target cell by regulating the entire gene expression pattern of the cell. By introducing the direct reprogramming induction factor into a cell and culturing the cell for a certain period of time, the cell can be induced to transform into a target cell having the gene expression pattern of a target type of cell. In the present invention, the "direct reprogramming induction factor" may be used interchangeably with "direct cell transformation induction factor," "cell transformation induction factor," and "reprogramming factor."

[0089] In the present invention, the term "introduction of a direct reprogramming induction factor" may refer to a method of administering a direct reprogramming induction factor to a culture medium of cells; a method of directly injecting a direct reprogramming induction factor into cells; a method of increasing or decreasing the expression level of a direct reprogramming induction factor present in cells; a method of transforming cells with an expression vector containing a gene encoding a direct reprogramming induction factor; a method of modifying a gene sequence so that the expression of a gene encoding a direct reprogramming induction factor is increased or decreased; a method of introducing an exogenous expression gene encoding a direct reprogramming induction factor; a method of treating a substance having an effect of inducing or inhibiting the expression of a direct reprogramming induction factor; and a combination thereof, but is not limited thereto, as long as the expression level of a direct reprogramming induction factor can be increased or decreased. In particular, the introduction of a direct reprogramming induction factor may induce the expression of a direct reprogramming induction factor under desired times and conditions. Specifically, the method for introducing the direct reprogramming induction factor into a cell may be, but is not limited to, a method of administering the direct reprogramming induction factor to a culture medium of cells, or a method of transforming cells with an expression vector including a gene encoding the direct reprogramming induction factor.

[0090] For example, the method of directly injecting the above-described direct reprogramming inducing factor into cells can be selected and used by any method known in the art, and is not limited thereto, but can be appropriately selected and applied from among methods using microinjection, electroporation, particle bombardment, direct muscle injection, insulator, and transposon.

[0091] In one specific example of the present invention, to compare the drNK of the present invention with the existing NK, Fresh primary NK (pNK) cells isolated from PBMC, pNK cells activated with IL-2 and IL-15 (ApNK) cells, or NK cell line (NK92, ATCC) were used as controls. According to the expression of CD56 and CD16 markers, the main cell populations were drNK: CD56 superbright CD16 superbright, NK92: CD56 bright CD16 dim , pNK (CD56 dim pNK): CD56 dim CD16 bright , ApNK (CD56 bright pNK): CD56 bright CD16 bright It was confirmed that NK cell phenotypes were distinguished, and in particular, CD56 was compared in the expression of NK cell receptors. dim Compared to pNK, it was confirmed that drNK had higher expression of NK cell activating receptors such as CD69, NKG2D, DNAM-1, and NKp46.

[0092]

[0093] In another specific example of the present invention, the gene expression of cytokines / chemokines expressed in the drNK of the present invention was compared with that of the control NK, and as a result, 10 types (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) that were overexpressed were confirmed, and 3 types of proteins (DDP4, M-CSF, BDNF) that were confirmed to be drNK-specific were identified.

[0094] In another specific example of the present invention, the correlation between the effect of natural killer cells on removing damaged nerve cells and the expression level of CD16 was confirmed, and the effect of each damaged nerve cell was confirmed by CD16 antibody. dim pNK (58.1%), CD56 brightIt was confirmed that the inhibition effect by CD16 antibody was proportional to the expression level of CD16, and in the case of drNK cells of the present invention, the inhibition effect by CD16 antibody was confirmed to be the greatest at 69.1%.

[0095] The term “neurological disease” of the present invention refers to a disease related to the nervous system, and is a disease that can be caused by damage, degeneration or loss of function of formed myelin (myelin sheath) or axons due to external or internal factors, loss or damage of nerve cells, etc.

[0096] In the present invention, the neurological disease specifically includes brain tumor, cerebral infarction, hypertensive cerebral hemorrhage, cerebral contusion, cerebral arteriovenous malformation, cerebral abscess, encephalitis, hydrocephalus, epilepsy, concussion, cerebral palsy, mild cognitive impairment, dementia, spinal cord tumor, spinal arteriovenous malformation, spinal cord infarction, pain, headache, migraine, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Batten disease, Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), MELAS syndrome (Mic acidosis and stroke-like episodes), MERRF syndrome (Myoclonic epilepsy with ragged-red fibers), NARP syndrome (Neurogenic weakness with ataxia and retinitis pigmentosa), Leigh syndrome, MIRAS syndrome (Mitochondrial recessive ataxia syndrome), degenerative neurological disease, schizophrenia, schizophreniform disorder, attention deficit hyperactivity disorder, personality disorder, Autism, post-traumatic stress disorder, anxiety disorder, panic disorder, depression, chronic stress-related depression, delusional disorder, obsessive-compulsive disorder, anorexia nervosa, bulimia nervosa, obesity, cerebral ischemic disease, neurodegenerative disease, diabetic neuropathy, traumatic nerve injury, neurodegenerative disease, neuropathic pain, epilepsy, chronic neuropathic pain, Guillain-Barré syndrome, myasthenia gravis, Rett syndrome, central sleep apnea, peripheral neuropathy, Charcot-Marie-Tooth disease, spinal muscular atrophy (SMA), autoimmune encephalitis, chronic traumatic encephalopathy (CTE), myotonic dystrophy dystrophy),May include, but is not limited to, multiple sclerosis, Schwannoma, Neurofibromatosis, Chronic inflammatory demyelinating polyneuropathy (CIDP), Polyneuropathy, and Neuroma.

[0097] The term "prevention" of the present invention refers to Schwann progenitor cells (SCPs) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2 and a combination thereof, or Schwann cells (SCs) expressing at least one selected from the group consisting of S100B, SOX10 and a combination thereof; and CD56. + , CD16 + It means any act of inhibiting or delaying the occurrence of a neurological disease by using a pharmaceutical composition for preventing or treating a neurological disease, which comprises an inducible natural killer (drNK) cell expressing at least one selected from the group consisting of: and a combination thereof.

[0098] The term "treatment" of the present invention refers to Schwann progenitor cells (SCPs) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2 and a combination thereof, or Schwann cells (SCs) expressing at least one selected from the group consisting of S100B, SOX10 and a combination thereof; and CD56. + , CD16 + It means any act of controlling or curing the symptoms of a neurological disease by using a pharmaceutical composition for preventing or treating a neurological disease, which comprises induced natural killer (drNK) cells expressing at least one selected from the group consisting of: and combinations thereof.

[0099] The term "pharmaceutical composition" of the present invention may include a pharmaceutically acceptable carrier, and may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

[0100] The pharmaceutically acceptable carrier may include, but is not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the pharmaceutical composition of the present invention may include diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants, and other pharmaceutically acceptable additives.

[0101] When the pharmaceutical composition of the present invention is formulated as an oral solid preparation, it may include tablets, pills, powders, granules, capsules, etc., and such solid preparation may include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may include a lubricant, such as magnesium stearate or talc, but is not limited thereto.

[0102] When the pharmaceutical composition of the present invention is formulated as an oral liquid, it may include a suspension, a solution, an emulsion, a syrup, etc., and may include a diluent such as water or liquid paraffin, a wetting agent, a sweetener, a fragrance, a preservative, etc., but is not limited thereto.

[0103] When the pharmaceutical composition of the present invention is formulated for parenteral use, it may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Non-aqueous solvents and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include, but are not limited to, Witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0104] In the present invention, the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, age, degree of disease, drug form, administration route, and period, but can be appropriately selected by a person skilled in the art.

[0105] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, for example, orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrauterinely, or intracerebrovascularly.

[0106] In one specific example of the present invention, as a result of confirming neurite growth by the SCP / SCP-SC and NK cells of the present invention, the SCP-SC only treatment group and the SCP-SC and drNK combination treatment group showed superior neurite growth compared to the control group, and in particular, it was confirmed that the SCP-SC and drNK combination treatment group was significantly higher than the SCP-SC only treatment group. In addition, in the partial nerve injury model, it was confirmed that the nerve growth effect was higher in SCP than in NCSC, and the nerve growth effect was significantly higher in the SCP and drNK combination treatment group than in the SCP only treatment group.

[0107] In one specific example of the present invention, when the composition of the present invention was transplanted into a sciatic nerve injury model mouse, it was confirmed that the average motor function was recovered better and the regenerative treatment effect was superior in the group that received a combination transplant of SCP and drNK compared to the group that received SCP alone.

[0108] This suggests that when induced Schwann progenitor cells (SCPs) and Schwann cells (SCs) differentiated therefrom, produced using the pluripotent stem cells (PSCs) of the present invention, are treated alone or in combination with natural killer cells in a model of nerve damage disease, the regeneration and treatment effects on the damaged nervous system are excellent.

[0109]

[0110] Another aspect of the present invention for achieving the above object is a Schwann precursor cell (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2 and a combination thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10 and a combination thereof; and CD56. + , CD16 + Provided is a cell therapy for the prevention or treatment of neurological diseases, comprising induced natural killer (drNK) cells expressing at least one selected from the group consisting of: and combinations thereof.

[0111] The terms used herein are as described above.

[0112] The term "cell therapy product" of the present invention refers to a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention by separating, culturing, and manufacturing cells and tissues from an individual through special manipulation, and by proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or by changing the biological characteristics of cells through other methods.

[0113] The above cell therapy composition may have an effect of preventing or treating a neurological disease by including Schwann precursor cells produced according to the method of the present invention or Schwann cells and induced natural killer cells differentiated therefrom.

[0114] The above cell therapy composition contains 1.0X10 to 1.0X10 of the Schwann precursor cells, Schwann cells, and induced natural killer cells based on the total weight of the composition. 10 Dog cells / ml, specifically 1.0X10 6 1.0X10 9 May include, but is not limited to, dog cells / ml.

[0115] The above cell therapy composition can be formulated into a unit dosage form pharmaceutical preparation suitable for administration into the patient's body according to a conventional method in the pharmaceutical field, and the preparation can contain an effective dosage amount through one or multiple administrations. Suitable dosage forms for this purpose include parenteral administration preparations such as injections such as injection ampoules, infusions such as infusion bags, and sprays such as aerosol preparations. The injection ampoules can be mixed and prepared with an injection solution immediately before use, and the injection solution can be physiological saline, glucose, mannitol, Ringer's solution, etc. In addition, the infusion bag can be made of polyvinyl chloride or polyethylene, and examples thereof include infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, and Terumo.

[0116] In addition to the active ingredient, the above pharmaceutical preparation may further include one or more pharmaceutically acceptable conventional inert carriers, for example, in the case of injections, a preservative, analgesic, solubilizer, or stabilizer, and in the case of topical administration preparations, a base, excipient, lubricant, or preservative.

[0117] The cell therapy composition of the present invention manufactured in this way or the pharmaceutical preparation thereof can be administered together with other cells used for the treatment of neurological diseases or in the form of a mixture with such cells using an administration method commonly used in the art, and specifically, it is possible to directly engraft or transplant into the diseased site of a patient requiring treatment, or directly transplant or inject into the abdominal cavity, but is not limited thereto. In addition, the administration is possible both non-surgically using a catheter and surgically, such as injection or transplantation after incision of the diseased site. In addition to parenterally administering, for example, directly to the lesion, transplantation by intravascular injection is also possible according to a conventional method.

[0118] The above cell therapy composition may be administered at a dosage of 0.0001 to 1,000 mg / kg per day, specifically 0.01 to 100 mg / kg, and the administration may be administered once a day or in several divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the above dosage does not limit the scope of the present invention in any way.

[0119]

[0120] One aspect of the present invention for achieving the above object provides a method for preventing or treating a neurological disease, comprising administering to a subject other than a human a suspected neurological disease a pharmaceutical composition for preventing or treating a neurological disease, the pharmaceutical composition comprising Schwann precursor cells (SCPs) or Schwann cells (SCs) differentiated therefrom as active ingredients; and natural killer (NK) cells.

[0121] Specifically, Schwann progenitor cells (SCPs) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof, or Schwann cells (SCs) expressing at least one selected from the group consisting of S100B, SOX10, and combinations thereof; and CD56. + , CD16 + A method for preventing or treating a neurological disease is provided, comprising administering to a subject other than a human a pharmaceutical composition for preventing or treating a neurological disease, the pharmaceutical composition comprising natural killer (NK) cells expressing at least one selected from the group consisting of: and combinations thereof; to a subject suspected of having a neurological disease.

[0122] The terms used herein are as described above.

[0123] The above "administration" means introducing the composition of the present invention into a subject by any suitable method, and the route of administration of the composition may be through any common route as long as it can reach the target tissue. It may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, or intranasal administration, but is not limited thereto.

[0124] The above "subject" refers to any animal, including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, excluding humans, that has or may develop a neurological disease. The type of subject is not limited as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.

[0125]

[0126] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and are not to be construed as limiting the scope of the present invention.

[0127]

[0128] Example 1. Differentiation of Schwann cell precursor (SCP) from human pluripotent stem cells (PSC)

[0129] A novel method that is an improvement on the existing production method (protocol 1 in Figure 1A) for differentiating pluripotent stem cells into Schwann progenitor cells was devised. To confirm the effects of FGF2, LDN193189, All-trans-retinoic acid (RA), StemRegenine1 (SR1), and Dorsomrphin as novel Schwann progenitor cell differentiation promoting candidates, the characteristics of cells differentiated by each of the eight methods of Portocol 1-8 were compared and analyzed, as shown in Figures 1A and 1B.

[0130] First, human pluripotent stem cells (PSCs), including human induced pluripotent stem cells derived from human neonatal foreskin fibroblasts (catalog number CRL-2097; ATCC) and human embryonic stem cells (H9 ESCs, WiCell), were cultured as follows. For feeder-free culture, cells were grown on Growth Factor-Reduced Matrigel (BD biosciences)-coated dishes in mTeSR1 medium (StemCell Technologies) with daily medium changes. To obtain human PSC-derived Schwann progenitor cells, colonized PSCs were replated onto Growth Factor-Reduced Matrigel-coated culture dishes. The following day, the culture medium was replaced with modified neural differentiation medium (NDM) containing SB431542 and CT99021 to neutralize the human PSC culture medium, and neural rosettes were formed by culturing for 6 days. Specifically, the NDM contained advanced DMEM / F12 and Neurobasal medium (1:1 mixture) containing 1x N2, 1x B27, 0.005% BSA, 2 mM Glutamax, 0.11 mM β-mercaptoethanol, 3 μM CT 99021 (Tocris Biosciences), 20 μM SB431542 (Tocris Biosciences), or additionally containing 20 μg / ml FGF2 (Peprotech), or 100 nM LDN193189 (Medchemexpress) in NDM medium as in Figures 1A and 1B.After 6 days of differentiation, the NDM medium was replaced with a neural induction medium (Schwann cell precursor induction medium (SCPDM)) containing 50 ng / ml NRG1, and the SCPDM medium contained 100 nM RA (all-trans retinoic acid, Sigma), 20 μg / ml FGF2, 2 μM SR1 (Stemregenin1, Cellagen), or 2 μM Dorsomorphin (Medchemexpress) depending on the experimental conditions, as shown in Fig. 1B. The SCPDM was replaced every 2 days, and when the cells reached 80% confluence, they were detached by treating with Accutase and cultured for an additional 6 days. In addition, the cells were proliferated by additional culture in SCPDM. After about 18 days of differentiation, in order to confirm whether SCP was created, the expression of SOX10, an SCP marker gene, was first confirmed through qPCR, and as a result, it was confirmed that it was highly expressed in Protocol 1, Protocol 4, Protocol 5, and Protocol 7 (Fig. 1C). Additionally, the expression levels of CD49d, ERBB3, and PLP1, known SCP marker genes, were confirmed through qPCR, and as a result, it was confirmed that they were highly expressed in Protocol 4 and Protocol 5, which are new differentiation methods, compared to the existing differentiation method of Protocol 1 (Fig. 1D). In particular, for ERBB3, the expression was found to be 2.7 times higher than before in Protocol 1 (average 2.15 times higher than GAPDH), Protocol 4 (average 5.99 times higher than GAPDH), and Protocol 5 (average 5.81 times higher than GAPDH), and for PLP1, the expression was found to be 2.7 times higher than before in Protocol 1 (average 7.39 times higher than GAPDH), Protocol 4 (average 17.2 times higher than GAPDH), and Protocol 5 (average 18.9 times higher than GAPDH).The expression was found to be more than 3 times higher. The protein expression of SOX10, GAP43, and IGFBP2 as Schwann progenitor cell marker proteins was confirmed through cell immunostaining in Protocol 1, Protocol 4, and Protocol 5 (Fig. 1E). SCPDM was used to induce and maintain PSC-SCP.

[0131]

[0132] Example 2. Production of Schwann Cell (SC) Differentiation from SCP

[0133] To confirm the Schwann cell differentiation potential of SCP differentiated by the new protocol, SC differentiation was performed together with existing iSCPs using the previously known SCP-SC differentiation method. To differentiate SCPs into Schwann cells, the SCPs and the SCPs produced by the somatic cell reprogramming technique (iSCPs) were cultured on Matrigel-coated plates in Schwann cell differentiation medium (SCDM). The SCDM contains DMEM containing 1% FBS, 200 ng / ml NRG1, 4 μM forskolin (Sigma), 100 nM all-trans retinoic acid (RA, Sigma), and 10 ng / ml PDGF-BB. After 3 days of culture, the culture medium was replaced with SCDM containing 1% FBS, 200 ng / ml NRG1, 10 ng / ml PDGF-BB (Thermo Fisher Scientific), but not forskolin or retinoic acid. After another 2 days, the culture medium was replaced with Schwann cell medium (SCM) containing 1% FBS and 200 ng / ml NRG1, but not forskolin, retinoic acid, or PDGF-BB. The cultured cells were maintained in SCM for expansion. Schwann cells were generated after 2 to 3 days of culture in SCM. On the 7th day of differentiation, the expression levels of SC marker genes S100b, NGFR, MPZ, and EGR2 were analyzed by qPCR, and no significant difference was observed compared to iSCP-SCs in which Schwann cell function was confirmed (Fig. 2A). Immunocytochemical analysis confirmed that most of the differentiated SCs were positive for S100B and SOX10, which are SC lineage-specific proteins (Fig. 2B). Therefore, it was confirmed that Schwann progenitor cells differentiated by the novel method successfully produced Schwann cells in a short period of time (approximately 7 days), similar to cells differentiated by the conventional method.

[0134]

[0135] Example 3. Confirmation of high neurotrophic factor GDNF, IGFBP-2 mRNA expression and protein secretion characteristics of induced SCP compared to conventional Schwann progenitor cells (NCSC).

[0136] In order to confirm the difference between the induced SCP of the present invention and NCSC, a representative Schwann precursor cell known to exist, in the protein secretion ability of cells, NCSC was first differentiated from human PSC (H9 ESC) using the following method.

[0137] Specifically, isolated PSCs were plated on Matrigel-coated culture dishes, and the next day, the culture medium was changed to DMEM supplemented with 1% Probumin (Millipore), 1% Penicillin-streptomycin, 1% L-alanyl-L-glutamine (Cellgro), 1% MEM non-essential amino acids, 0.1% Trace elements A (Cellgro), 0.1% Trace elements B (Cellgro), 0.1% Trace elements C (Cellgro), 0.11 mM β-mercaptoethanol, 10 μg / ml Transferrin, 50 μg / ml (+)-sodium l-ascorbate (Sigma), 10 ng / ml NRG1 (Peprotech), 200 ng / ml LONG R3 IGF-I (Sigma), 3 μM BIO (Tocris Biosciences), 20 μM SB431542 (Tocris Biosciences). and replaced with NCSC induction medium (NCSCIM) containing 8 ng / ml FGF2 (Peprotech). The culture medium was changed daily. NCSCs were produced after approximately 20 days of NCSCIM growth. Unless otherwise specified, all reagents were purchased from Thermo Fisher Scientific.

[0138] The expression levels of neurotrophic factors GDNF and IGFBP-2 were quantitatively analyzed using qPCR from hPSCs, the differentiated NCSCs, and the cells of the induced SCP of the present invention, and the results confirmed that the expression levels were significantly higher in SCPs than in NCSCs (Fig. 3A). In order to obtain a conditioned medium (CM) for comparative analysis of proteins secreted from NCSCs and induced SCPs that affect nerve regeneration and growth, 10 5 SCP cells and NCSC cells were seeded in 30 mm culture dishes with 2 ml of culture medium. After 48 hours, the cultured medium was filtered using a 0.22 μm filter (Millipore). To measure the concentration of the secreted neurotrophic factor GDNF, ELISA was performed in conditioned medium derived from SCP and NCSC according to the manufacturer's protocol (Abcam).

[0139] As a result, it was confirmed that more was secreted in SCP (16.6 pg / ml) compared to NCSC (3.3 pg / ml) (left side of Fig. 3B). To measure cytokine levels in NCSC-CM and SCP-CM, a proteome profiler array (Proteome Profiler Human XL Cytokine Array Kit, ARY022B; R&D systems) was used according to the manufacturer's instructions. The results of the above example were obtained by acquiring images through Amersham imager 600 (GE Healthcare Life Sciences) and then quantitatively analyzed using ImageJ (Open source software). As a result, it was confirmed that more was secreted in SCP (1150.5 MPD) compared to NCSC [157 MPD (Mean Pixel Density)] (right side of Fig. 3B). Therefore, it can be inferred that the nerve growth factor secreted in high levels in SCP of the present invention compared to NCSC can have a greater effect on nerve growth and regeneration.

[0140]

[0141] Example 4. Phenotypic Characterization of Induced Natural Killer (drNK) Cells

[0142] To obtain induced drNK cells, reprogramming factor OSKM was introduced into PBMCs separated from human peripheral blood by Ficoll gradient, and then PBMC cells and polybrene (4 μg / ml) were cultured together for 1 day. The next day, 3x10 5 The transformed cells were cultured for an additional 5 days in culture medium RIM (StemSpan SFEM II containing 10% FBS, 1% Penicillin / Streptomycin, 5 μM CHIR99021, 20 ng / ml Human IL-3, 20 ng / ml Human IL-6, 20 ng / ml Human SCF, 20 ng / ml Human FLT3L, 20 ng / ml Human TPO). The transformed cells were cultured for 18-40 days in culture medium RMM (StemSpan SFEM II containing 10% FBS, 1% Penicillin / Streptomycin, 200 IU / ml Human IL-2, 20 ng / ml Human IL-7, 20 ng / ml Human IL-15, 20 ng / ml Human SCF, 20 ng / ml Human FLT3L, 2 μM StemRegenin I).

[0143] To confirm whether drNK cells were produced through the above direct reprogramming, the cells were stained with anti-CD56-APC (Biolegend) antibody and anti-CD16-PE (Biolegend) antibody as markers of NK cells, and then NK cells (CD56+ and CD16) were analyzed using flow cytometry. +) group was analyzed. Fresh primary NK (pNK) cells isolated from PBMCs, pNK (ApNK) cells activated with 200 IU / ml Human IL-2 and 20 ng / ml Human IL-15 for 4–14 days, and NK cell line (NK92, ATCC) were used as control cells.

[0144] According to the expression of CD56 and CD16 markers, the main cell populations are drNK: CD56superbrightCD16superbright, NK92: CD56 bright CD16 dim , pNK (CD56 dim pNK): CD56 dim CD16 bright , ApNK (CD56 bright pNK): CD56 bright CD16 bright It was confirmed that NK cell phenotypes were distinguished (Figures 4A and 4B). Here, the most commonly used pNK (CD56 dim Based on the intensity of CD56 and CD16 fluorescence of pNK (dim (10 4 below), bright (10 4 -10 5 ), superbright (10 5 (above) was expressed as follows. In particular, in the comparison of NK cell receptor expression, CD56 dim Compared to pNK, we confirmed that drNK had higher expression of NK cell activating receptors such as CD69, NKG2D, DNAM-1, and NKp46 (Fig. 4C).

[0145]

[0146] Example 5. Quantitative analysis of drNK expression cytokine / chemokine gene levels

[0147] To produce iPSC-NK cells as a control for comparison with drNK, iPSCs were dissociated into single cells by ReLeSR (Stem Cell Technologies) treatment and added to STEMdiff APEL2 medium (Stem Cell Technologies), a medium composition for spindle embryoid bodies, containing 1x penicillin / streptomycin (Invitrogen), 40 ng / ml SCF (Invitrogen), 20 ng / ml VEGF (R&D), and 20 ng / ml BMP4 (R&D), and 3 x 10 4Cells suspended in cells / ml were seeded at 3,000 cells per well in a round-bottom 96-well plate, centrifuged at 1,500 rpm for 4 minutes at 8°C, and cultured in a 37°C incubator for 3 to 4 days. Afterwards, half of the culture medium was replaced with new culture medium and cultured for 9 to 11 days. On days 9 to 11 of embryoid body differentiation, embryoid bodies from 6 to 8 wells of a 96-well plate were transferred to 1 well of a 24-well plate coated with 2% Gelatin, and the differentiation culture medium was 85% DMEM / F12 (GIBCO), 15% FBS (GIBCO), 5 ng / ml sodium selenite (Sigma), 50 μM ethanolamine (Sigma), 20 μg / ml ascorbic acid (Sigma), 25 μM β-mercaptoethanol (GIBCO), 1x Glutamax (GIBCO), 1% penicillin / streptomycin (GIBCO), and 5 ng / ml IL-3 (Peprotech), 10 ng / ml IL-15 (Peprotech), 20 ng / ml IL-7 (Peprotech), 20 ng / ml SCF (Invitrogen), 10 ng / ml Flt3L. (Peprotech) cytokines were added, and the cells were cultured for 28 days in NK differentiation medium with cytokines except IL-3 added every 5 to 7 days. The differentiated iPSC-NK cells were isolated using an NK isolation kit (Miltenyi Biotec) and then cultured in a culture medium containing 90% RPMI 1640, 10% FBS, 1% penicillin / streptomycin, 20 ng / ml IL-15, and 20 ng / ml IL-2.

[0148] qRT-PCR was performed to quantitatively analyze the gene expression of cytokines / chemokines expressed in the above drNK compared to control NK (NK-92 and iPS-NK cells).

[0149] As a result, it was confirmed that expression in drNK was relatively increased compared to the NK-92 and iPS-NK groups. When the amount expressed in NK-92 is set to 1, the fold expression in iPS-NK and drNK is expressed as [CCL5: NK-92 (1), iPS-NK (0.85), drNK (3.64), IFNr: NK-92 (1), iPS-NK (0.1), drNK (1.9), CXCL11: NK-92 (1), iPS-NK (0.01), drNK (2.93), CXCL12: NK-92 (1), iPS-NK (1.82), drNK (5.73), GDNF: NK-92 (1), iPS-NK (0.03), drNK (5.69), VEGF: NK-92 (1), iPS-NK (1.29), drNK (2.3), XCL1: NK-92 (1), iPS-NK (0.18), drNK (2.15), IL16: NK-92 (1), iPS-NK (0.98), drNK (4.35); LIF: NK-92 (1), iPS-NK (1.78), drNK (8.5); LTB: NK-92 (1), iPS-NK (1.02), drNK (2.01)] (Fig. 5). Therefore, as a result of comparative analysis of the expression levels of cytokine / chemokine molecular target genes that may play an important role in NK function in NK-92, iPS-NK, and drNK, 10 types (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) overexpressed in drNK were identified.

[0150]

[0151] Example 6. Identification of drNK secretion factors using human proteome cytokine arrays.

[0152] We analyzed the secretory substances, especially cytokine secretion, that can affect the neural regeneration of drNK cells in culture media. Specifically, to obtain conditioned medium (CM) of NK cells, drNK cells and CD56 dim 10 pNK cells in a culture dish 6 The cells were cultured at a density of 10 cells / ml. After 24 hours, the cultured medium was filtered using a 0.22 μm filter (Millipore). drNK-CM and CD56 dim To measure cytokine levels in pNK-CM, a proteome profiler array (Proteome Profiler Human XL Cytokine Array Kit, ARY022B; R&D Systems) was used according to the manufacturer's instructions. ImageJ software was used for quantitative analysis of the final images.

[0153] As a result, 56 drNK secreted proteins (RANTES (average = 81,174 MPD), DPPIV (average = 78,046 MPD), CD31 (average = 33,901 MPD), TIM-3 (average = 31,688 MPD), Emmprin (average = 29,204 MPD), MIP-1α / MIP-1ß (average = 27,937 MPD), GM-CSF (average = 22,299 MPD), Fas Ligand (average = 21,602 MPD), MIF (average = 19,398 MPD), IL-16 (average = 16,867 MPD), IL-17A (average = 8,249 MPD), Flt-3 Ligand (average = 5834 MPD), ICAM-1) were identified through ImageJ (image intensity = 1,000 Mean Pixel Density (MPD) or higher). (average=4,532 MPD), M-CSF (average=2,867 MPD), FGF-19 (average=2,481 MPD), Serpin E1, MIP-3ß (average=2,165 MPD), IL-18 (average=2,144 MPD), IL-32 (average=1,950 MPD), Angiogenin (average=1,901 MPD), IL-1α (average=1,895 MPD), Cystatin C (average=1,792 MPD), Resistin (average=1,699 MPD), GDF-15 (average=1,635 MPD), Angiopoietin-2 (average=1,558 MPD), TNF-α (average=1,546 MPD), PDGF-AA (average=1,536 MPD), Apolipoprotein AI (average=1,507 MPD), Osteopontin (average=1,489 MPD), Dkk-1 (average=1,447 MPD), uPAR (average=1,422 MPD), Endoglin (average=1,411 MPD), IFN-γ (average=1,394 MPD), FGF basic (average=1,381 MPD), SDF-1α (CXCL12) (average=1,378 MPD), IL-1ß (average=1,363 MPD), RAGE (average=1,327 MPD), EGF (average=1,277 MPD),IL-8 (average=1,252 MPD), IL-27 (average=1,208 MPD), BAFF (average=1,191 MPD), CD40 ligand (average=1,159 MPD), IL-34 (average=1,129 MPD), VCAM-1 (average=1,127 MPD), IL-1ra (average=1,113 MPD), MCP-1 (average=1,094 MPD), Kallikrein 3 (average=1,088 MPD), IL-12 p70 (average=1,088 MPD), IL-11 (average=1,087 MPD), ST2 (average=1,059 MPD), MMP-9 (average=1,058 MPD), IL-22 (average=1,057 MPD), C-Reactive Protein (average=1,041 MPD), BDNF (average=1,034 MPD), Vitamin D BP (mean=1,032 MPD), Lipocalin-2 (mean=1,004 MPD) were identified (Fig. 6A). Control group CD56, dim28 proteins secreted at quantitatively higher levels than pNK [IL-16 (5.58 times), BAFF (5.23 times), CD31 (4.36 times), ICAM-1 (3.57 times), Emmprin (3.39 times), VCAM-1 (3.33 times), Flt-3 Ligand (3.32 times), Cystatin C (3.16 times), C-Reactive Protein (2.58 times), IL-27 (2.44 times), TNF-a (2.22 times), IL-32 (2.16 times), TIM-3 (2.01 times), IL-12 p70 (1.94 times), uPAR (1.53 times), IL-18 Bpa (1.40 times), MIF (1.39 times), Dkk-1 (1.35 times), IL-11 (1.33 times), GM-CSF (1.30 times), RANTES (1.29 fold), Endoglin (1.24 fold), IL-22 (1.16 fold), RAGE, Osteopontin (1.06 fold), GDF-15 (1.02 fold), Kallikrein 3 (1.02 fold), Angiopoietin-2 (1.02 fold)] were identified (Fig. 6B). Three proteins (DDP4, M-CSF, BDNF) that were identified as drNK-specific were identified (Fig. 6C).

[0154]

[0155] Example 7. Confirmation of drNK's effect on clearing damaged nerves and verification of its dependence on CD16 expression.

[0156] To confirm the selective removal of damaged neurons by NK cells, a partially damaged SH-SY5Y model was used. To induce damaged neurons, SH-SY5Y cells were treated with DMEM / F12 (1:1) medium containing 10% FBS and 200 μM H2O2 for 24 h, and it was confirmed that most cells were labeled with ROS-labeled (MitoSox Red, Thermofisher Scientific), indicating damaged cells (Fig. 7A). To induce partially damaged neurons, SH-SY5Y cells were treated with DMEM / F12 (1:1) medium containing 10% FBS and 200 μM H2O2 for 24 h. Cells that were not treated with H2O2 and SH-SY5Y cells that were treated were mixed at a ratio of 1:3 and plated the next day. CD56 dim The number of ROS-positive neurons was confirmed 24 hours after co-culture with pNK and drNK (E:T=0.25:1). Compared to the ratio of ROS-positive neurons in the control group that was not co-cultured with NK cells (56.5%), CD56 dim Not only was it confirmed that pNK (22.0%) and drNK (8.9%) were significantly reduced, but also CD56 dim It was confirmed that more than twice as many damaged nerve cells were removed by drNK compared to pNK (Figure 7B).

[0157] To confirm the correlation between the effect of natural killer cells on removing damaged neurons and the expression level of CD16, SH-SY5Y neurons, the damaged neurons model of the above example, were treated with H2O2 to induce neural damage, and then co-cultured (E:T=1:1) with CD16 antibody and NK cells for 4 hours. The effect of NK cells on removing damaged neurons was analyzed. Specifically, SH-SY5Y neurons were treated with 200 nM H2O2 in DMEM / F12 (1:1) medium containing 10% FBS for 24 hours to induce neural damage. Cell tracker-labeled NK cells, damaged neurons, and CD16 antibody (Biolegend, 1:20 concentration used) were cultured alone or co-cultured (E:T=1:1), and after 4 hours, Propidium iodide (PI, Thermofisher Scientific) staining and flow cytometry were performed to confirm the results (Fig. 7C).

[0158] As a result, assuming the effect of removing damaged neurons when treated alone without antibodies as the standard (100%), the effect of removing damaged neurons by CD16 antibody was CD56. dim pNK (58.1%), CD56 bright It was confirmed that pNK (50.1%), NK92 (82.6%), and drNK (30.9%) were reduced. In conclusion, it was confirmed that the inhibitory effect by CD16 antibody was proportional to the expression level of CD16, and in the case of drNK cells of the present invention, the inhibitory effect by CD16 antibody was confirmed to be the greatest at 69.1% (Fig. 7D). The list of antibodies used in the present invention is as follows in Table 1.

[0159] ProteinCompany (Cat. No.)DilutionSOX10Abcam (AB155279)1:200GAP43Abcam (AB75810)1:500TUJ1Abcam (AB78078)1:1000IGFBP2Cell signaling Technology (3922)1:200S100BAbcam (ab52642)1:1000MBPAbcam (ab209328)1:1000CD16Biolegend (302008)1:20CD56Biolegend (318310)1:20CD69Biolegend (310906)1:20CD94Biolegend (305508)1:20NKG2DBiolegend (320808)1:20NKp46Biolegend (331908)1:20DNAM-1Biolegend (338312)1:20KIR2DL2 / 3Biolegend (312604)1:20KIR3DL1Biolegend (312706)1:20

[0160] Example 8. Confirmation of neurite outgrowth by SCP / SCP-SC and NK cells

[0161] To confirm neurite outgrowth by SCP / SCP-SC and NK cells, a scratch model of neurons differentiated from hiPSCs and a partially damaged SH-SY5Y model were used (Fig. 8A). First, iPSCs were cultured in embryoid body (EB) culture medium (90% DMEM / F12 and 10% Serum replacement containing 1% Penicillin / Streptomycin, 1X MEM NEAA, 1X Glutamax, 0.1 mM β-mercaptoethanol) with a colony size of 500 x 500 μm for 7 days in suspension, and then cultured in neurosphere (NS) culture medium (DMEM / F12 and 1X N2, 1X B27, 1% Penicillin / Streptomycin, 20 ng / ml hEGF, 20 ng / ml bFGF, 10 ng / ml hLIF) with a rotating orbital rotation speed of 20 to 22 rpm. NS cells were passaged every 5 to 7 days to a size of 250 x 250 μm, and at the second passage (NS p2), NS p2 was attached to a cover glass coated with 0.01% PLL (Poly-L-lysine) at 4°C for 24 hours. The cells were cultured in the attached NS culture medium (Neurobasal medium (Cat. no. 21103-049, GIBCO) and 1X N2, 1X B27, 1% Penicillin / Streptomycin, 1X Glutamax, 25 ng / ml BDNF, 25 ng / ml GDNF) for 7 to 14 days, and damage experiments were performed when neurites extended from the NS. Damaged neurites extended from the NS body were removed (damaged) by scraping with the tip of a sterilized pipette tip, and the next day after damage, SC (10,000 cells / cm) was cultured. 2) and drNK (10,000 cells / ml) were co-cultured. Cell morphology and βIII tubulin antibody (Cat. no. ab78078, abcam, 1:200 dilution) were immunohistochemically stained after 24 hours of co-culture. Images before and after injury and immunostaining 24 hours after co-culture of SCP-SC (GFP labeled) and drNK showed neurites (TUJ1: red) and cell nuclei (DAPI: blue), confirming the difference in neurite growth in each experimental group (Fig. 8B).

[0162] In the same manner as in Example 8B, neurite outgrowth was quantitatively analyzed after 48 hours of culture following SCP-SC treatment alone and SCP-SC+drNK combination treatment. As a result, compared to the control group (average length = 157.7 μm), the SCP-SC alone treatment group (average length = 251.7 μm) and the SCP-SC and drNK combination treatment group (average length = 315.9 μm) showed significantly greater neurite outgrowth (Fig. 8C). In particular, it was confirmed that the SCP-SC and drNK combination treatment group was 1.25 times higher on average than the SCP-SC alone treatment group.

[0163] To confirm neurite outgrowth by SCP / SCP-SC and NK cells in a partial nerve injury model, SH-SY5Y cells were treated for 24 hours in DMEM / F12 (1:1) medium containing 10% FBS and 200 μM H2O2 as in Example 7B above, and SH-SY5Y cells that were not treated with H2O2 and treated were mixed in a 1:3 ratio and plated on a Matrigel-coated plate with differentiation medium (DMEM / F12 (1:1) medium containing 1 μM Retinoic acid and 2% FBS) at a density of 2x10 3 cells / cm 2After seeding, the differentiated SH-SY5Y cells were cultured in a 5% CO2 incubator for 2 days. After 2 days of culture, the differentiated SH-SY5Y cells were treated with NK, SCP, SCP-SC, NCSC or NGF (Peprotech) [ratio = 2 (neuronal cells): 1] and cultured for an additional 2 days, and then neurite outgrowth was analyzed. Images were acquired using an Axio Vert.A1 microscope (Carl Zeiss). The length of neurites in each image was measured using AxioVs40 v4.8.2.0 software. As a result, compared to the control group (average length = 43.5 μm), the SCP-SC treatment group (average length = 72.9 μm) and CD56 dim pNK treatment group (mean length = 57.4 μm), drNK treatment group (mean length = 59.8 μm), SCP-SC and CD56 dim Neurites in the pNK-treated group (mean length = 87.3 μm), SCP-SC and drNK-treated groups (mean length = 158.6 μm), NGF-treated group (mean length = 60.1 μm), and NGF and drNK-treated groups (mean length = 67.2 μm), pSC-treated group (mean length = 45.8 μm), pSC and CD56 dim Growth was confirmed to be promoted in the pNK treatment group (average length = 57.1 μm), pSC and drNK treatment groups (average length = 65.1 μm) (Fig. 8D). Here, compared to pSC, SCP-SC had an average of 1.59 times more CD56 dim It was confirmed that drNK had a significantly higher nerve growth effect by an average of 1.04 times compared to pNK, and by an average of 2.17 times compared to the SCP-SC alone treatment group in the SCP-SC and drNK combination treatment group.

[0164] In the case of combined treatment with SCP and NCSC, the SCP treatment group (mean length = 71.1 μm), the SCP and drNK treatment group (mean length = 81.7 μm), and the SCP and CD56 dimpNK treatment group (mean length = 53.7 μm), NCSC treatment group (mean length = 53.7 μm), NCSC and drNK treatment group (mean length = 67.3 μm), NCSC and CD56 dim pNK treatment group (average length = 62.5 μm) was confirmed (Fig. 8E). Therefore, it was confirmed that the nerve growth effect was significantly higher in the SCP group by an average of 1.32 times compared to the NCSC group, and in the SCP and drNK combination treatment group by an average of 1.14 times compared to the SCP alone treatment group.

[0165]

[0166] Example 9. Promotion of nerve regeneration and therapeutic effects by SCP and NK cell transplantation alone or in combination in an animal model of sciatic nerve injury.

[0167] To analyze the nerve recovery and therapeutic effects by in vivo Schwann cell precursors, natural killer cells alone, and combined transplantation, a sciatic nerve injury mouse model was used (Fig. 9A). First, the left sciatic nerve of 8-week-old C57BL / 6 male mice was damaged by cutting the central region, creating a 2-3 mm nerve defect. Schwann cell precursors differentiated in Example 1 and NCSCs differentiated in Example 3 were cultured on Matrigel (Matrigel, 2 x 10 4 cells / μl) and diluted to 1 x 10 5 Cells (5 μl of cell suspension containing SCP labeled with GFP by lentiviral infection) were transplanted into the neural defect site. Along with this, the induced natural killer (drNK) cells (1 x 10 7 cells / 150 μl) were transplanted into the tail vein (Fig. 9A). Sixteen weeks after transplantation, the sciatic nerves of each group were sampled and immunohistochemically analyzed to confirm the promotion of nerve regeneration and therapeutic effects of NCSC cell-only transplantation and SCP combined transplantation.

[0168] As a result, it was confirmed through immunohistochemical analysis that MBP-positive nerve cell regeneration was superior in the SCP-transplanted group compared to the NCSC-transplanted group, and the MBP-positive nerve cell ratio was measured through quantitative analysis and confirmed to be approximately 2.1 times higher in the SCP-transplanted group [control group NCSC (80.6 units / 0.2 mm 2 ), SCP of the present invention (195.3 units / 0.2 mm 2 )] (Fig. 9C).

[0169] In the above sciatic nerve injury model, a Rotarod test was conducted to verify the effects of NCSC, SCP, and SCP and NK cell complex transplantation on motor ability 8 weeks later. As a result, it was confirmed that the group transplanted with the SCP of the present invention showed an approximately 1.45-fold improvement in motor ability compared to the control group NCSC [control group NCSC (55.6 sec), SCP of the present invention (80.94 sec)] (Fig. 9D).

[0170] In the same way, the average motor function recovery of the group that received a combined transplant of SCP and drNK compared to the SCP-only group was analyzed through the Rotarod test, and it was confirmed that the treatment effect was approximately 1.26 times better in the case of combined transplant of SCP and drNK [control group: SCP-only (100 sec), drNK (89.0 sec), SCP+drNK (126.6 sec)] (Fig. 9E).

[0171]

[0172] Example 10. Promotion of nerve regeneration and therapeutic effect by SCP-SC and NK cell transplantation alone or in combination.

[0173] A partial sciatic nerve injury model was used to analyze the nerve recovery and therapeutic effects by single or combined transplantation of nerve growth factor (NGF), Schwann cells, and natural killer cells. First, partial sciatic nerve injury was induced by pinching the central region of the sciatic nerve of 8-week-old immunodeficient male Balb / c-nude mice with forceps. The injured area was seeded with Matrigel (Matrigel, 2 x 10) containing PBS or SCP-SCs cells (Peprotech) containing 20 μg / ml NGF. 4 Each 5 μl of the cell suspension prepared by diluting 10 cells / μl was transplanted into the nerve defect area. In addition, as shown in Figure 10A, CD56 bright pNK or drNK cells (1 x 10 7 cells) were transplanted into the tail vein. Four weeks after transplantation, the sciatic nerves of each group were sampled and the efficacy of peripheral nerve disease treatment was confirmed by morphological analysis using hematoxylin and eosin (H&E) staining. Figure 10A shows the morphological analysis of each group (NGF, CD56 bright pNK, drNK, SCP-SC, CD56 bright These are representative staining results according to pNK+SCP-SC, drNK+SCP-SC. Morphologically, the existing CD56 in the single treatment group dim Compared to pNK, drNK of the present invention demonstrates superior nerve recovery and regenerative effects. In addition, it was confirmed that the SCP-SC+NK complex treatment group exhibited a relatively superior regenerative treatment effect compared to the group treated alone (Fig. 10A).

[0174] As a result of immunohistochemical analysis in samples such as the above Figure 10A, cells positive for the neuronal cell marker TUJ1 were identified, and compared to the existing control drug NGF, SCP-SC and CD56 bright It was confirmed that the pNK and drNK single treatment groups showed a significantly and relatively high nerve regeneration promotion effect [NGF (8.0%), SCP-SC (28.6%), CD56bright pNK (16.3%), drNK (25%)]. Here, the existing CD56 bright Compared to pNK, the drNK of the present invention demonstrated a 1.53-fold superior neural recovery and regenerative effect. The SCP-SC+drNK combination treatment group demonstrated the most effective regenerative therapeutic effect compared to single transplantation. In particular, CD56 bright It was confirmed that the drNK+SCP-SC complex treatment group showed an average 1.98 times higher nerve regeneration effect than the pNK+SCP-SC complex treatment group [SCP-SC+drNK (47.6%), CD56 bright pNK+SCP-SC (24.0%)] (Figs. 10 B and 10C).

[0175] To analyze the transplantation status and myelination degree in the drNK, SCP-SC, and drNK+SCP-SC groups in the sciatic nerve model confirmed in the above Example 10, immunohistochemical analysis and qRT-PCR analysis of the Schwann cell marker S100 and the myelination marker MBP were performed 4 weeks after transplantation. As a result, it was confirmed that in the regenerated area where drNK+SCP-SC was composite-transplanted, S100 (Figs. 11 A and 11B) and MBP (Figs. 11C and 11D) protein and gene expression [S100, Control (1), drNK (0.99), SCP-SC (1.38), drNK+SCP-SC (2.24); MBP, Control (1), drNK (1.09), SCP-SC (1.6), drNK+SCP-SC (2.2)] were significantly increased compared to the single transplantation group. In particular, when comparing the expression levels of myelin-positive genes, it was confirmed that the combined treatment group had expression levels approximately 1.37 times higher than the SCP-SC single treatment group.

[0176] In the above Example 10, the recovery of motor function according to nerve regeneration after sciatic nerve injury was tested using a Rotarod test 4 weeks after transplantation. The SCP-SC transplant group showed the best motor function recovery effect among the single transplantation groups (an average of 1.25 times higher than NGF), and CD56 bright We confirmed that the motor function recovery effect of the drNK transplant group was superior to that of pNK [NGF (46 sec), CD56 bright pNK (42 sec), drNK (54 sec), SCP-SC (57.7 sec)]. In addition, among the experimental groups, the drNK+SCP-SC composite transplantation group showed the highest improvement in motor function, while the control group, CD56 bright The pNK+SCP-SC composite transplantation group showed relatively low efficacy [drNK+SCP-SC (68.4 sec), CD56 bright pNK+SCP-SC (53.3 sec)]. In particular, compared to the SCP-SC transplantation group, which showed the greatest effect in the single transplantation group, the SCP-SC and drNK combined transplantation group showed an average motor function recovery effect that was 1.18 times higher with statistical significance (Fig. 12).

[0177]

[0178] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A pharmaceutical composition for the prevention or treatment of neurological diseases, comprising Schwann precursor cells (SCP) or Schwann cells (SC) differentiated therefrom; and natural killer (NK) cells as active ingredients, The above Schwann progenitor cells (SCPs) express at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof; The above Schwann cells (SC) express at least one selected from the group consisting of S100B, SOX10 and combinations thereof; and The above natural killer (NK) cells are CD56 + , CD16 + and expressing at least one selected from the group consisting of combinations thereof; A pharmaceutical composition for the prevention or treatment of neurological diseases.

2. In the first paragraph, the Schwann precursor cell (SCP) is (a) a step of culturing pluripotent stem cells in a first medium containing SB431542 and CT99021; and (b) a step of culturing the cells cultured in step (a) with a second medium to which NRG1 (Neuregulin-1) is additionally added, A pharmaceutical composition for preventing or treating a neurological disease, which is prepared by a method for preparing Schwann progenitor cells from pluripotent stem cells.

3. A pharmaceutical composition for preventing or treating a neurological disease, wherein in the first paragraph, the first medium of step (a) may additionally contain FGF2, and the second medium of step (b) may additionally contain stemlegenin I.

4. In the first paragraph, the Schwann cell (SC) is (a) a step of culturing pluripotent stem cells in a first medium containing SB431542 and CT99021; and (b) a step of culturing the cells cultured in step (a) using a second medium to which NRG1 (Neuregulin-1) is additionally added; (c) a step of recovering Schwann progenitor cells from the cultured medium; and (d) a step of culturing the recovered Schwann progenitor cells in a third medium containing FBS and NRG1; A pharmaceutical composition for preventing or treating a neurological disease, which is prepared by a method for preparing Schwann cells from pluripotent stem cells.

5. A pharmaceutical composition for preventing or treating a neurological disease, wherein in the fourth paragraph, the third medium of step (d) further comprises at least one selected from the group consisting of retinoic acid, forskolin, and PDGF-BB.

6. In the first paragraph, the natural killer (NK) cell is CD56 dim , CD56 bright , CD56 superbright , CD16 dim , CD16 bright , CD16 superbright A pharmaceutical composition for preventing or treating a neurological disease, wherein the composition expresses at least one compound selected from the group consisting of: and combinations thereof.

7. In paragraph 1, the natural killer (NK) cell is (a) a step of introducing a reprogramming factor into the separated cell; (b) a step of culturing the cells of step (a) from the next day after the introduction of the reprogramming factor in a first medium containing i) cytokines, growth factors, and GSK3β (Glycogen synthase kinase 3β) inhibitors to increase the efficiency of direct reprogramming, and ii) a step of culturing the cells in a second medium containing cytokines, growth factors, and AHR (Aryl hydrocarbon receptor) inhibitors to promote the production of natural killer cells. A pharmaceutical composition for preventing or treating a neurological disease, which is prepared by a method for preparing induced natural killer cells (drNK) from isolated cells.

8. A pharmaceutical composition for preventing or treating a neurological disease, wherein the induced natural killer (drNK) cells in paragraph 7 overexpress at least one gene selected from the group consisting of CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, and LTB, compared to a control group.

9. A pharmaceutical composition for preventing or treating a neurological disease, wherein the induced natural killer (drNK) cells in paragraph 6 express at least one protein selected from the group consisting of DDP4, M-CSF, and BDNF.

10. In any one of paragraphs 1 to 8, the neurological disease is brain tumor, cerebral infarction, hypertensive cerebral hemorrhage, brain contusion, cerebral arteriovenous malformation, brain abscess, encephalitis, hydrocephalus, epilepsy, concussion, cerebral palsy, mild cognitive impairment, dementia, spinal tumor, spinal arteriovenous malformation, spinal cord infarction, pain, headache, migraine, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Batten disease, Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), MELAS syndrome (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), MERRF syndrome (Myoclonic epilepsy with ragged-red fibers), NARP syndrome (Neurogenic weakness with ataxia and retinitis pigmentosa), Leigh syndrome, MIRAS syndrome (Mitochondrial recessive ataxia). syndrome), neurodegenerative diseases, schizophrenia, schizophreniform disorder, attention deficit hyperactivity disorder (ADHD), personality disorders, autism, post-traumatic stress disorder (PTSD), anxiety disorders, panic disorders, depression, chronic stress-related depression, delusional disorders, obsessive-compulsive disorders, anorexia nervosa, bulimia nervosa, obesity, cerebral ischemic diseases, neurodegenerative diseases, diabetic neuropathy, traumatic nerve injury, neurodegenerative diseases, neuropathic pain, epilepsy, chronic neuropathic pain, Guillain-Barre syndrome, Myasthenia gravis, Rett syndrome, central sleep apnea, peripheral neuropathy, Charcot-Marie-Tooth disease, spinal muscular atrophy (SMA), autoimmune encephalitis,A pharmaceutical composition for preventing or treating a neurological disease, characterized in that the disease is at least one disease selected from the group consisting of Chronic traumatic encephalopathy (CTE), Myotonic dystrophy, Multiple sclerosis, Schwannoma, Neurofibromatosis, Chronic inflammatory demyelinating polyneuropathy (CIDP), Polyneuropathy, and Neuroma.

11. A cell therapy agent for preventing or treating a neurological disease, comprising as an active ingredient Schwann precursor cells (SCP) or Schwann cells (SC) differentiated therefrom; and induced natural killer (drNK) cells, wherein the Schwann precursor cells (SCP) express at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and a combination thereof; The above Schwann cells (SC) express at least one selected from the group consisting of S100B, SOX10 and combinations thereof; and The above natural killer (NK) cells are CD56 + , CD16 + and expressing at least one selected from the group consisting of combinations thereof; Cell therapy agent for preventing or treating neurological diseases.

12. In paragraph 11, the natural killer (NK) cell is CD56 dim , CD56 bright , CD56 superbright , CD16 dim , CD16 bright , CD16 superbright A cell therapy agent for preventing or treating a neurological disease, which expresses at least one selected from the group consisting of: and combinations thereof.

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