A composition for the prevention or treatment of neurological diseases, comprising Schwann cell precursor (SCP) or Schwann cells differentiated therefrom (SC), and natural killer cells (NK) cells.

Abstract

The present invention relates to a pharmaceutical composition and cell therapy agent for the prevention or treatment of neurological diseases, comprising Schwann cell precursors (SCPs) or Schwann cells differentiated therefrom, derived from pluripotent stem cells (PSCs) or somatic cells, and natural killer (NK) cells as active ingredients. Specifically, the Schwann cell precursors (SCPs) express at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof; the Schwann cells (SCs) express at least one selected from the group consisting of S100B, SOX10, and combinations thereof; and the natural killer (NK) cells express CD56 + CD16 + It is characterized by expressing at least one selected from the group consisting of the and combinations thereof.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition and cell therapy agent for the prevention or treatment of neurological diseases, comprising Schwann cell precursors (SCPs) or Schwann cells differentiated therefrom, derived from pluripotent stem cells (PSCs) or somatic cells, and natural killer (NK) cells as active ingredients. Specifically, the Schwann cell precursors (SCPs) express at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof; the Schwann cells (SCs) express at least one selected from the group consisting of S100B, SOX10, and combinations thereof; and the natural killer (NK) cells express CD56 + CD16 + It is characterized by expressing at least one selected from the group consisting of the and combinations thereof. [Background technology]

[0002] Schwann cells (SCs) are essential glial cells in the peripheral nervous system (PNS) that play a central role in supporting neurons and promoting nerve repair. In the PNS, Schwann cells are responsible for forming myelin, which insulates nerve fibers and ensures the rapid transmission of nerve impulses. Myelination by Schwann cells is crucial not only for normal nerve function but also for the repair and regeneration of damaged nerves. In addition to their role in myelin formation, 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), supporting nerve survival, axon growth enhancement, and post-injury nerve regeneration. Furthermore, Schwann cells generate extracellular matrix components that provide support for axon guidance and regrowth, creating an environment conducive to nerve repair.

[0003] The unique characteristics of human Schwann cells are extremely useful for disease modeling, drug discovery, and the development of new therapies such as cell therapy, but securing a sufficient number of cells that retain their functionality is difficult. In primary cultured Schwann cells derived from the human body, the isolation efficiency and culture purity vary greatly depending on the nerve biopsy conditions, and collection requires invasive procedures that carry risks such as pain and potential nerve damage. In in vitro culture, Schwann cells have low proliferative capacity, making it difficult to expand to a sufficient number of cells, and as culture time progresses, the purity and quality of Schwann cells decrease due to increased contamination by fibroblasts. It is important that cultured Schwann cells retain their original characteristics (myelination ability, neurotrophic factor secretion, etc.), but long-term culture and expansion in vitro leads to the loss of the characteristics and functions of Schwann cells.

[0004] As an alternative, research and development are actively underway to optimize and expand the production of Schwann cells from stem cells. In particular, human pluripotent stem cells (PSCs), which include human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs), are attracting attention as an important resource for the differentiation and production of Schwann cells due to their excellent proliferative and differentiation capabilities. Throughout the developmental process, Schwann cells exist in various forms, including 1) neural crest (stem) cells (NC(S)Cs), Schwann cell precursors (SCPs), 2) immature Schwann cells without myelin formation, and 3) mature Schwann cells with myelin sheaths. Generally, a method used to obtain Schwann cells from PSCs involves first differentiating neural crest stem cells (NCSCs), which are developmental precursors of Schwann cells and possess multipotency, and then redifferentiating the NCSCs into Schwann cells. However, this method of differentiating PSCs into Schwann cells via NCSCs has several problems: 1) the differentiation process is complex and time-consuming, 2) productivity and purity are low, and 3) biological function and therapeutic performance are low.

[0005] In contrast, Schwann cell precursors (SCPs) are an intermediate cell type that exists separately from neural crest cells, which appear in the early stages of development, and Schwann cells, which are in the pre-myelin formation stage. Schwann cell precursors (SCPs) obtained from PSCs can be cultured and are attracting attention as an optimal Schwann cell source for direct production of Schwann cells in a short period of time. Therefore, this technology produces Schwann cell precursors (PSC-SCPs) and Schwann cells (PSC-SCP-SCs) from PSCs and analyzes their effects in the treatment of nerve injury / diseases.

[0006] On the other hand, natural killer (NK) cells are a type of lymphoid blood cell that plays a crucial role in both innate and acquired immune responses. In particular, they do not recognize specific antigens, but sense abnormal proteins on the cell surface or a decrease in major histocompatibility complex (MHC) I molecules. They recognize and immediately eliminate disease-causing abnormal cells such as cancer cells and infected cells of viruses, bacteria, fungi, and parasites, making them an important target for the development of therapeutic agents for various diseases.

[0007] NK cells promote nerve recovery / regeneration by removing damaged nerve cells, influence other immune cells such as microglia and T cells, suppress the induction of neuroinflammation and autoimmune diseases, and remove nerve cells infected with viruses, thereby suppressing the spread of infection within nerve tissue. Therefore, the potential usefulness of NK cells in the treatment of various neurological diseases, such as nerve injury diseases and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, is attracting attention. However, research and technological development regarding their direct roles and potential therapeutic effects are currently insufficient.

[0008] In recent years, with the advancement of direct reprogramming technology, there has been a surge in the development of techniques to directly produce highly clinically useful functional cells without going through the stem cell production process, such as PSCs. Functional cells produced by direct reprogramming technology have a low risk of epigenetic remodeling and tumor formation, and the simplification of the cell production process makes it easy to improve safety, reliability, and efficiency, which are highly valued technical advantages. These characteristics are expected to ultimately dramatically shorten and reduce the time and cost required for the development of therapeutic agents, and contribute to the elimination of barriers to practical application. Therefore, research and development efforts are continuing to secure raw materials for cell therapies targeting various diseases. Accordingly, this technology analyzes the effects of Schwann cell precursors (drSCP) and NK (drNK) cells obtained by direct reprogramming in the treatment of nerve injury / disease.

[0009] To date, there have been no reports on the preventive, therapeutic, or ameliorative effects of combining NK cells with Schwann cell precursors (PSC-SCP) derived from pluripotent stem cells (PSC-SCP-SC), or Schwann cell precursors (drSCP) or Schwann cell precursors (drSCP-SC) induced by somatic cell reprogramming, on neurological diseases. [Overview of the project] [Problems that the invention aims to solve]

[0010] The inventors, in their efforts to develop a method for rapidly producing nerve injury / disease treatment agents with improved functionality and high production efficiency, have found that by producing Schwann cell precursors that can be proliferated in vitro through differentiation culture from human pluripotent stem cells or direct reprogramming culture from somatic cells, and by inducing differentiation from these Schwann cell precursors into Schwann cells, it is possible to produce human Schwann cells with improved functionality both in vivo and ex vivo in a shortened time and under improved production efficiency conditions. Furthermore, they have confirmed that combining these with human natural killer (NK) cells is useful for the prevention or treatment of neurological diseases, thus completing the present invention. [Means for solving the problem]

[0011] The present invention relates to a Schwann cell precursor (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10, and combinations thereof, and CD56 + CD16 + The objective is to provide a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising natural killer (NK) cells expressing at least one selected from the group consisting of combinations thereof.

[0012] Furthermore, the present invention relates to a Schwann cell precursor (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2 and combinations thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10 and combinations thereof, and CD56 + CD16 + The objective is to provide a cell therapy composition for the prevention or treatment of neurological diseases, comprising natural killer (NK) cells expressing at least one selected from the group consisting of combinations thereof.

[0013] Furthermore, the present invention provides a method for preventing or treating a neurological disease, which includes administering a pharmaceutical composition for preventing or treating a neurological disease, the composition containing Schwann cell precursors (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 natural killer (NK) cells expressing at least one selected from the group consisting of CD56 + , CD16 + to an individual suspected of having a neurological disease other than human.

Advantages of the Invention

[0014] When treating peripheral and central nervous system injury disease models with Schwann cell precursors (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 natural killer (NK) cells expressing at least one selected from the group consisting of CD56 + , CD16 + alone or in combination, they show gene expression characteristics different from those of conventional Schwann cell precursors (NCSCs), have an excellent ability to secrete the neurotrophic factor GDNF compared to conventional NCSCs, and have been confirmed to have excellent effects on the regeneration and treatment of damaged nervous systems. Therefore, they are useful for preventing or treating neurological diseases.

Brief Description of the Drawings

[0015] [Figure 1]Figure 1A shows a schematic diagram of the process of differentiating human pluripotent stem cells (hPSCs) into Schwann cell precursors (hSCPs), and shows the development results of Protocols 4 and 5, which are improved by the introduction of new factors compared to the conventional control group Protocol 1. Figure 1A shows schematic diagrams of the differentiation of human pluripotent stem cells (hPSCs) into Schwann cell precursors (hSCPs) in the conventional control group (Protocol 1) and the present invention (Protocols 2-8). Figure 1B shows the composition of various differentiation media used in Stages 1 and 2 of Figure 1A. Figure 1C shows the results of quantitative analysis of the expression level of the SOX10 gene from total RNA of cells on day 24 using the differentiation induction method of Figure 1B. Figure 1D shows the results of further quantitative analysis of the gene expression levels of CD49d, ERBB3, and PLP1 in the group in which an increase in SOX10 was confirmed in protocols 1, 4, 5, and 7 of Figure 1C. Figure 1E shows the results of confirming the protein expression of SOX10, GAP43, and IGFBP2 in SCPs differentiated by the conventional method (protocol 1) and the novel method of introducing new factors (Protocols 4 and 5) by immunocytochemistry. [Figure 2] Figure 2A shows the results of quantitative analysis of the gene expression of Schwann cell markers S100b, NGFR, MPZ, and EGR2 in Schwann cells differentiated from Schwann cell precursors produced by Protocols 1 and 4 in Figure 1 (Protocol 1-SC, Protocol 4-SC), and Schwann cells produced by differentiation induction culture from SCPs (iSCP) produced by somatic cell reprogramming (iSCP-SC). Figure 2B shows the results of confirming the protein expression of SOX10 and S100B in the differentiated Schwann cells from Figure 2A. [Figure 3]Figure 3A shows the results of confirming that the expression of neurotrophic factors GDNF and IGFBP-2 is higher in the induced SCP of the present invention compared to conventional Schwann cell precursors (NCSCs). Figure 3B shows the results of quantitative analysis of the gene expression levels of GDNF and IGFBP-2 in H9 (hPSC), NCSC, and SCP, respectively. [Figure 4] Figure 4A shows the cellular phenotypic characteristics of the specified induced natural killer (drNK) cells of the present invention. Figure 4A lists four representative NK cell types (1. CD56dimpNK, the main cell type of PBMC-derived NK cells; 2. CD56brightpNK, 3. CD56brightpNK, 4. CD56dimpNK, 5. CD56brightpNK, 6. CD56brightpNK, 7. CD56brightpNK, 8. CD56brightpNK, 9. CD56brightpNK, 10. 11. 11. 12. 12. 13. 14. 15. 11. 12. 12. 13. 14. 12. 13. 14. 15. 14. 15. 14. 15. 14. 15. 14. 15. 16. 14. 15. 14. 15. 16. 14. 15. 16. 17. 14. 15. 16. 17. 189. 14. 15. 16. 17. 18. 19. 14. 15. 16. 17. 18. 19. 18. 19. 14. 15. 16. 17. 18. 19. 18. 19. 14. 18. 19. 14. 15. 16. 17. 18. 19. 18. 19. [Figure 5] This figure shows the results of quantitative analysis of cytokine gene expression levels expressed in drNK by qRT-PCR. It identifies 10 cytokines (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) that are expressed at higher levels in drNK compared to the control groups NK-92 and iPS-NK. [Figure 6]Figure 6A shows the results of identifying various CD56dimpNK and drNK cells in their respective conditioning media using human proteome cytokine sequences. Figure 6B shows the results of quantitative analysis of 56 secreted proteins in the drNK cell conditioning media. Figure 6C shows the results of quantitative analysis of 28 secreted proteins that increased in the drNK cell conditioning media compared to the CD56dimpNK cell conditioning media. Figure 6C shows the results of identifying DPP4, M-CSF, and BDNF, proteins specifically secreted in the drNK cell conditioning media. [Figure 7] Figure 7A shows the results of confirming the nerve clearing effect of drNK and verifying its dependence on CD16 expression. Figure 7B shows the results of confirming the significantly superior nerve clearing effect of drNK compared to the control group under co-culture conditions of NK cells and ROS-positive injured nerve cells. Figure 7C is a schematic diagram of a cytotoxicity assay using an anti-CD16 antibody. Figure 7D shows the results of analyzing the relationship between the nerve clearing effect and CD16 expression. The results show that the clearing effect was most greatly affected by the CD16 antibody in drNK cells with the highest CD16 expression. Therefore, the results confirm that the clearing activity of drNK cells is related to CD16 expression. [Figure 8]Figure 8A shows the results of confirming significantly superior effects compared to the control group in neurite outgrowth by SCP / SCP-SC and drNK cells. Figure 8A is a schematic diagram of the nerve recovery / regeneration assay using NGF treatment of a nerve regeneration control group substance, SCP, SC, NCSC, and NK cells alone, and co-culture in an axonal transection or partial nerve injury model. Figure 8B shows the results of confirming the recovery / regeneration effect of neurite length after neurite transection injury in stem cell-derived nerve cells using drNK, SCP-SC, drNK+SCP-SC alone, and co-culture, using cell phase contrast microscopy images and images positive for the nerve cell marker TUJ1. This figure shows the results of obtaining not only the nerve regeneration promoting effect of drNK and SCP-SC, but also the synergistic effect of nerve regeneration by drNK+SCP-SC. Figure 8C shows the results of a comparative analysis of the recovery / regeneration effects of SCP-differentiated SCs (SCP-SCs) and drNK of the present invention, both individually and in combination, on neurite damage in a partially injured nerve cell model, using NGF, CD56dimpNK, and primary cultured Schwann cells (pSCs) as control groups. Figure 8D shows the results of a comparative analysis of the recovery / regeneration effects of conventional control group SCP (NCSCs) and the present invention's SCP, and conventional control group CD56dimpNK and the present invention's drNK on neurite damage. [Figure 9] Figure 9A shows the results of confirming the promotion of sciatic nerve regeneration and therapeutic effects by transplantation of SCP and NK cells alone or in combination in an animal model. Figure 9B and C 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. Figure 9D shows the results of analyzing motor function recovery by nerve regeneration using the Rotarod test in the animal model of Figure 10A, confirming that motor function was significantly improved in the group transplanted with the SCP of the present invention compared to the control group NCSC. Figure 9E shows the results of obtaining a significantly superior nerve regeneration effect with combined SCP+drNK treatment compared to treatment alone. [Figure 10]Figure 10A shows the results of confirming the nerve regeneration promotion and therapeutic effects of SCP-SC and NK cell transplantation alone or in combination in an animal model of partial sciatic nerve injury. In the sciatic nerve injury model, sciatic nerves were sampled from each group four weeks after transplantation of NGF, CD56brightpNK, drNK, and SCP-SC cells alone or in combination. The therapeutic effect on peripheral nerve disease was confirmed by hematoxylin-eosin staining (H&E). In the group treated alone, it was confirmed that drNK of the present invention had a superior nerve recovery / regeneration effect compared to conventional CD56brightNK, and that SCP-SC+N was superior to the group treated alone. This figure shows the results confirming that the K-compound treatment group obtained a relatively superior regenerative therapeutic effect. Figures 10B and 10C show the results of quantitative analysis of positive images of the neuronal cell marker TUJ1 immunohistochemical staining to confirm the therapeutic effect after conventional control drug NGF treatment, NK, SCP-SC alone, and combined transplantation. The results show that the SCP-SC, CD56brightpNK, and drNK alone treatment groups obtained a significantly greater nerve regeneration promoting effect compared to the conventional control drug NGF, and that the drNK of the present invention is superior to conventional CD56brightpNK in nerve recovery / regeneration effect. [Figure 11]Figure 10 shows the results of confirming the promotion of nerve regeneration and therapeutic effects by transplantation of SCP-SC and NK cells alone or in combination in an animal model. Figure 11A shows the results of immunostaining for the Schwann cell marker S100 in nerve bundles at the site of injury. Figure 11B shows the results of quantitative analysis of S100 gene expression in total mRNA obtained from nerve bundles at the site of injury. Figure 11C shows the results of immunostaining for the myelin marker MBP in nerve bundles at the site of injury. Figure 11D shows the results of quantitative analysis of MBP gene expression in total mRNA obtained from nerve bundles at the site of injury. Myelin marker MBP immunohistochemical staining and qRT-PCR analysis confirmed that the SCP-SC + drNK combined transplantation group showed the largest increase in S100 and MBP-positive cells compared to the SCP-SC + drNK cell alone transplantation group. Therefore, it was confirmed that myelin sheath regeneration was most improved in the SCP-SC + drNK combined transplantation group, and that this was contributed to improved nerve function. [Figure 12] Figure 10 shows the results of behavioral experiments (rotarod) in an animal model, confirming the nerve regeneration promotion and therapeutic effects of SCP-SC and NK cell transplantation alone or in combination. In the group with only SCP-SC transplantation, the SCP-SC transplantation group showed the best improvement in motor function, and the drNK transplantation group showed superior improvement in motor function compared to CD56brightpNK. In the comparison group, the drNK + SCP-SC combined transplantation group showed the greatest improvement in motor function. [Modes for carrying out the invention]

[0016] These will be explained in detail below. Note that each description and embodiment disclosed in this invention applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this invention is included. Furthermore, this invention is not limited to the following specific descriptions.

[0017] To achieve the above objective, one aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising Schwann cell precursors (SCPs) or Schwann cells differentiated therefrom (SCs), and natural killer (NK) cells as active ingredients.

[0018] Specifically, a Schwann cell precursor (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10, and combinations thereof, and CD56 + CD16 + The present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising natural killer (NK) cells expressing at least one selected from the group consisting of combinations thereof.

[0019] The inventors were the first to discover that treating peripheral and central nervous system injury models with the above composition resulted in gene expression characteristics different from conventional Schwann cell precursors (NCSCs), superior gamma-dendritic neurotrophic factor (GDNF) secretion capacity compared to conventional NCSCs, and superior regenerative and therapeutic effects on damaged nervous systems.

[0020] In particular, the Schwann cell precursor (SCP) of the present invention was found to have significantly higher expression levels of the neurotrophic factors GDNF and IGFBP-2 genes, and higher secretion levels of proteins that affect nerve regeneration and growth, compared to NCSC, a representative Schwann cell precursor known conventionally.

[0021] Furthermore, since the induced natural killer (drNK) cells of the present invention express higher levels of NK cell activation receptors such as CD69, NKG2D, DNAM-1, and NKp46 compared to control NK cells, we identified 10 cytokine / chemokine genes that are overexpressed in the drNK cells of the present invention and confirmed three proteins that are specifically found only in drNK cells.

[0022] Furthermore, when peripheral and central nervous system injury disease models were treated with a combination of Schwann cell precursors (SCPs) or Schwann cells differentiated therefrom (SCs) and natural killer (NK) cells, it was confirmed that the combined SCP-SC and NK treatment group exhibited superior nerve extension and regenerative therapeutic effects compared to the single-treatment group.

[0023] In particular, when the induced natural killer (drNK) cells of the present invention were used to treat peripheral and central nervous system injury disease models, the combined SCP-SC and NK treatment group showed relatively superior nerve extension and regenerative therapeutic effects compared to the control group NK or the single-treatment group.

[0024] This suggests that the composition of the present invention is useful for the prevention or treatment of neurological diseases because, compared to conventionally known SCPs or NK cells, it not only exhibits higher levels of gene expression or protein secretion that affect nerve regeneration and growth, but also shows superior nerve growth and regeneration effects in combined treatment compared to treatment with them alone.

[0025] As a specific example, the Schwann cell precursor (SCP) of the present invention is prepared by a method for producing SCP from PSCs, which includes (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) has been further added. However, the method is not limited to this.

[0026] In this invention, "SB431542" is a specific repressor of TGF-β (Transforming growth factor-β) and has the structure of chemical formula 1.

[0027] [ka]

[0028] Specifically, SB431542 is contained in concentrations of 1 to 100 μM, more specifically 1 to 50 μM, even more specifically 1 to 30 μM, and even more specifically 5 to 25 μM, but is not limited to these concentrations.

[0029] In this invention, "CT99021" and CHIR-99021 (CT99021) are GSK-3α / β repressors, and are also referred to as CT99021, CHIR99021, CHIR 99021, CHIR-99021, or CT-99021. They have the structure of chemical formula 2.

[0030] [ka]

[0031] Specifically, the CT99021 is contained in concentrations of 1 to 100 μM, more specifically 1 to 50 μM, even more specifically 1 to 10 μM, and even more specifically 1 to 5 μM, but is not limited to these concentrations.

[0032] In this invention, "NRG1 (Neuregulin-1)" refers to the protein encoded by the NRG1 gene, which acts on the EGFR receptor. Specifically, the NRG1 is contained in concentrations of 1 to 1000 ng / ml, more specifically 10 to 500 ng / ml, even more specifically 20 to 200 ng / ml, and even more specifically 30 to 100 ng / ml, but is not limited to these concentrations.

[0033] Other specific examples include, but are not limited to, a first medium in step (a) further containing FGF2, and a second medium in step (b) further containing StemRegenin I (SR I).

[0034] In this invention, "FGF2 (Fibroblast growth factor 2)" is a fibroblast growth factor and is used in combination with bFGF (basic fibroblast growth factor) or FGF-β. Specifically, the FGF2 is contained at concentrations of 1 to 100 μg / ml, more specifically 1 to 50 μg / ml, even more specifically 5 to 50 μg / ml, and even more specifically 10 to 30 μg / ml, but is not limited to these concentrations.

[0035] In the present invention, "StemRegenin I (SR I)" means an aryl hydrocarbon receptor inhibitor, specifically 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol hydrochloride. StemRegenin I may further be contained in the second medium for producing Schwann cell precursors (SCPs). SR I is contained at concentrations 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 to these concentrations.

[0036] Another specific aspect of the present invention provides Schwann cell precursors (SCPs) produced by the method described above. The Schwann cell precursors produced by the method of the present invention are Schwann cell precursors differentiated from pluripotent stem cells (PSCs), and these Schwann cell precursors have the potential to differentiate into Schwann cells, melanocytes, etc., a high proliferation rate (expandability), and to be maintained for a long period of time. Furthermore, they express at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof, which are Schwann cell precursor-specific marker genes.

[0037] In this invention, "Schwann cell precursor (SCP)" refers to an intermediate stage in the neural crest development process that Schwann cells pass through. Specifically, it is an intermediate cell between neural crest (stem) cells (NC(S)C) and immature Schwann cells before myelin formation. The Schwann cell precursor can differentiate into Schwann cells.

[0038] In this invention, "pluripotent stem cells (PSCs)" refers to undifferentiated stem cells that have the ability to differentiate into all three germ layers (endoderm, mesoderm, and ectoderm). Under in vitro culture conditions, undifferentiated pluripotent stem cells maintain a normal karyotype and possess pluripotency and self-renewal ability. In this invention, pluripotency includes both differentiated pluripotency and multipotency. Pluripotent stem cells that are differentiated include embryonic carcinoma (EC) cells, embryonic stem (ES) cells, and embryonic germ (EG) cells. Specifically, the pluripotent stem cells of this invention are human-derived ES cells (hESCs) or induced pluripotent stem (iPSC) cells (hiPSCs), but any species from which they originate may be used as long as they possess pluripotency.

[0039] In one specific example of the present invention, when the differences between the SCP of the present invention and neural crest stem cells (NCSCs), which are representative Schwann cell precursors known conventionally, were examined, it was confirmed that the expression levels of the neurotrophic factors GDNF and IGFBP-2 genes were significantly higher in the SCP compared to NCSCs, and that the secretion levels of proteins that affect nerve regeneration and growth were higher. Therefore, it can be seen that the nerve growth factors secreted in greater quantities in the SCP of the present invention compared to conventional NCSCs can provide an even greater effect on nerve growth and regeneration.

[0040] As yet another specific example, the Schwann cells derived from human pluripotent stem cells-Schwann cell precursors (PSC-SCP-SC) of the present invention may be produced by a method for producing Schwann cells from PSCs, which includes 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) has been further added, (c) recovering SCP from the culture medium, and (d) culturing the recovered SCP in a third medium containing FBS and NRG1.

[0041] As another specific example, the third medium in step (d) above further contains, but is not limited to, at least one selected from the group consisting of retinoic acid, forskolin, and PDGF-BB.

[0042] In this invention, "retinoic acid" refers to a metabolite produced when vitamin A is broken down in the body, and C 20 H 28 It has the chemical formula O2. It is known to have effects such as suppressing colorectal cancer and treating rheumatoid arthritis. Specifically, the retinoic acid is contained in the culture medium at concentrations of 1 to 300 nM, more specifically 10 to 200 nM, and even more specifically 50 to 150 nM, but is not limited to these.

[0043] In this invention, "forskolin" refers to labdane diterpene produced from the Indian Coleus plant (Plectranthus barbatus). Specifically, the forskolin is contained in the culture medium at concentrations of 1 to 100 μM, more specifically 1 to 50 μM, and even more specifically 1 to 10 μM, but is not limited to these concentrations.

[0044] In this invention, "PDGF-BB (Platelet-derived growth factor-BB)" refers to a dimer (homoidimer) of PDGFB encoded by the PDGFB gene. Specifically, the PDGF-BB is contained in the culture medium at concentrations 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 to these concentrations.

[0045] In this invention, "Schwann cells (SC)" are glial cells in the peripheral nervous system that play roles such as myelin formation, nerve impulse transmission, and neurotrophic factor secretion, and are known to particularly influence nerve survival and axon growth.

[0046] A further aspect of the present invention provides Schwann cells produced by the method described above. Schwann cells produced by the method of the present invention are Schwann cells produced from pluripotent stem cells via Schwann cell precursors, and exhibit positive expression of Schwann cell-specific marker genes such as S100B and SOX10.

[0047] As another specific example, the aforementioned natural killer (NK) cells are CD56 + CD16 + The cells express at least one selected from the group consisting of CD56 and combinations thereof, but are not limited thereto. Specifically, the natural killer (NK) cells express CD56 dim CD56 bright CD56 superbright CD16 dim CD16 bright CD16 superbright It expresses, but is not limited to, at least one selected from the group consisting of combinations thereof.

[0048] For example, (1) pNK isolated from human peripheral blood, (2) CD56 isolated from human peripheral blood dim CD16 bright(2) CD56 obtained by activating pNK isolated from human peripheral blood with IL-2 / IL-15 cytokines. bright CD16 bright pNK, (3) immortalized CD56 bright CD16 dim This refers to NK cell lines (NK92), but is not limited to these.

[0049] As another specific example, the directly reprogrammed NK (drNK) cells may be produced by a method for producing directly reprogrammed NK cells from isolated cells, which includes the steps of (a) introducing a reprogramming factor into isolated cells, and (b) starting the day after the introduction of the reprogramming factor, culturing the cells from step (a) in a first medium containing cytokines, growth factors, and a GSK3β (Glycogen synthase kinase 3β) inhibitor to improve the efficiency of direct reprogramming, and ii) culturing them in a second medium containing cytokines, growth factors, and an AHR (Aryl hydrocarbon receptor) inhibitor to promote the production of directly reprogrammed NK (drNK) cells.

[0050] A further specific aspect of the present invention provides induced natural killer cells (drNK) produced by the method described above. Induced natural killer cells produced by the method of the present invention are CD56 superbright CD16 superbright It expresses, but is not limited to, at least one selected from the group consisting of combinations thereof.

[0051] As another specific example, the induced natural killer (drNK) cells described above 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 the control group, but are not limited to this.

[0052] As another specific example, the induced natural killer (drNK) cells express at least one protein selected from the group consisting of DPP4, M-CSF, and BDNF, but are not limited to this.

[0053] In this invention, "cytokines" refers to various relatively small proteins produced by cells and used in cell signaling, and affect cells containing them. Generally, they are related to, but are not limited to, immune responses to inflammation or infection. Specifically, the cytokines 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.

[0054] In the present invention, "growth factor" refers to polypeptides that promote the 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).

[0055] For the purposes of the present invention, cytokines and growth factors are contained in a culture medium for directly reprogramming isolated cells into converted cells, and any type of cytokine and growth factor is acceptable as long as they are used for direct reprogramming.

[0056] In this invention, "natural killer (NK) cells" refer to core innate immune cells that immediately recognize and eliminate infections caused by viruses, bacteria, fungi, and parasites, as well as abnormal self-cells. Unlike T cells, which recognize target cells by expressing antigen-specific receptors, NK cells do not have specificity for antigens and do not perform human leukocyte antigen (HLA) matching. Instead, they recognize abnormal changes in target cells, such as the balance of inhibitory or activating receptors like killer immunoglobulin receptors (KIR), natural cytotoxicity receptors (NCR), DNAM-1 (DNAX accessory molecule-1), and NKG2D (NK group 2 member D), the disappearance of surface MHC (Major histocompatibility complex) class I antigens, and the accumulation of abnormal proteins, and exhibit contact-dependent cytotoxicity through various mechanisms. Unlike T cells, which cause graft-versus-host disease (GVHD) in non-self allogeneic cells with mismatched human leukocyte antigens (HLA), allogeneic NK cells have been shown to have very few side effects of graft-versus-host disease and, in fact, exhibit strong therapeutic effects.

[0057] In this invention, "direct reprogramming" refers to a method of converting a specific cell lineage into a target cell with completely different characteristics by adjusting the global gene expression pattern of that cell. Direct reprogramming is a concept that includes, but is not limited to, cell reprogramming, differentiation, direct differentiation, dedifferentiation, direct dedifferentiation, conversion, direct conversion, trans-differentiation, and direct trans-differentiation.

[0058] The aforementioned direct reprogramming may involve introducing an oligonucleotide or vector containing an exogenous gene or DNA into a cell to perform "cell transformation," or it may involve changing the cell into a different state. The aforementioned "differentiation" refers to the phenomenon in which daughter cells produced by cell division have functions different from those of the original mother cell. In the present invention, the terms "direct reprogramming" are used interchangeably with "direct cell transformation induction," "direct cell transformation," and "cell transformation."

[0059] For the purposes of this invention, natural killer cells are obtained by direct reprogramming and are used in combination with induced natural killer (drNK) cells.

[0060] In this invention, "isolated cells" are not particularly limited and include cells whose lineage has already been identified, such as germ cells, somatic cells, and progenitor cells. "Somatic cells" refers to all differentiated cells that make up plants and animals, excluding germ cells, and "progenitor cells" refers to mother cells that do not express specific differentiation traits but have the fate of differentiation, in cases where cells corresponding to offspring express specific differentiation traits. For example, in blood cells, hematopoietic stem cells correspond to progenitor cells, and in mesenchymal cells, mesenchymal stem cells correspond to progenitor cells.

[0061] The isolated cells are, but are not limited to, human-derived cells; cells from various individuals are included in this invention. Furthermore, the isolated cells of this invention include all in vivo and in vivo cells.

[0062] For example, the isolated cells are somatic cells; for other examples, somatic cells excluding NK cells; and for yet another example, at least one selected from the group consisting of blood cells and fibroblasts, but not limited to these. For example, the blood cells are peripheral blood mononuclear cells (PBMCs), but not limited to these.

[0063] In this invention, "direct reprogramming inducer" refers to a gene (or polynucleotide) introduced into a cell to induce cell transformation, or a protein encoded by such gene. The direct reprogramming inducer varies depending on the target cell to be obtained through reprogramming and the type of cell before transformation. Cell transformation using the direct reprogramming inducer induces transformation into a target cell by regulating the entire gene expression pattern of the cell. By introducing the direct reprogramming inducer into a cell and culturing the cell for a predetermined period, it is possible to induce cell transformation into a target cell having the gene expression pattern of the desired type of cell. In this invention, the term "direct reprogramming inducer" is used interchangeably with "direct cell transformation inducer," "cell transformation inducer," and "reprogramming factor."

[0064] The "introduction of direct reprogramming inducers" in the present invention is carried out by methods such as administering the direct reprogramming inducer to a cell culture medium, directly injecting the direct reprogramming inducer into cells, increasing or decreasing the expression level of the direct reprogramming inducer present in cells, transforming cells with an expression vector containing a gene encoding the direct reprogramming inducer, modifying the gene sequence so that the expression of the gene encoding the direct reprogramming inducer increases or decreases, introducing an exogenous gene encoding the direct reprogramming inducer, treating cells with a substance that has an expression-inducing or expression-inhibiting effect on the direct reprogramming inducer, and combining these methods to increase or decrease the expression level of the direct reprogramming inducer in cells. However, the invention is not limited to the above examples as long as it increases or decreases the expression level of the direct reprogramming inducer. In particular, the introduction of the direct reprogramming inducer may involve inducing the expression of the direct reprogramming inducer for a desired time and under desired conditions. Specifically, the methods for introducing the direct reprogramming inducer into cells include administering the direct reprogramming inducer to a cell culture medium and transforming cells with an expression vector containing a gene encoding the direct reprogramming inducer, but are not limited to these.

[0065] For example, the method for directly injecting the direct reprogramming inducer into cells can be any method known in the art, and is not limited to these, but can be appropriately selected and applied from methods such as microinjection, electroporation, particle bombardment, direct intramuscular injection, insulator, and transposon.

[0066] In one specific example of the present invention, to compare the drNK of the present invention with conventional NK, fresh primary NK (pNK) cells isolated from PBMCs, pNK (ApNK) cells activated with IL-2 and IL-15, or NK cell lines (NK92, ATCC) were used as the control group. Depending on the expression of each CD56 and CD16 marker, the major cell group was 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 can be divided into those categories, and in particular, when comparing the expression of the NK cell receptor, CD56 dim Compared to pNK cells, drNK cells were found to express higher levels of NK cell activation receptors such as CD69, NKG2D, DNAM-1, and NKp46.

[0067] In another specific example of the present invention, when the gene expression of cytokines / chemokines expressed in drNK of the present invention was compared with that of a control group NK, 10 overexpressed cytokines (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) were identified, and three proteins specifically identified in drNK (DPP4, M-CSF, BDNF) were identified.

[0068] In another specific example of the present invention, when the correlation between the effect of natural killer cells in removing damaged nerve cells and the expression level of CD16 was confirmed, it was found that the effect of CD16 antibodies on removing each type of damaged nerve cell was CD56. dim pNK (58.1%), CD56 brightA decrease in pNK (50.1%), NK92 (82.6%), and drNK (30.9%) was confirmed. In conclusion, it was confirmed that the inhibitory effect of the CD16 antibody is proportional to the expression level of CD16, and in the drNK cells of the present invention, the inhibitory effect of the CD16 antibody was 69.1%, confirming that it has the greatest inhibitory effect.

[0069] In this invention, "neurological disease" refers to a disease related to the nervous system, and is a disease caused by external or internal factors such as damage, regression, or loss of function of formed myelin (myelin sheath) or axons, or loss or damage of nerve cells.

[0070] In this invention, neurological diseases specifically include brain tumors, cerebral infarction, hypertensive cerebral hemorrhage, cerebral contusion, arteriovenous malformation, brain abscess, encephalitis, chickenpox, epilepsy, concussion, cerebral palsy, mild cognitive impairment, dementia, spinal cord tumors, spinal arteriovenous malformations, spinal cord infarction, pain, headache, migraine, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Button's disease, Kearns-Sayre syndrome (KSS), chronic progressive extraocular muscle palsy (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), Reye's syndrome, and MIRAS syndrome (Mitochondrial recessive ataxia). Syndrome, degenerative neurological disorders, schizophrenia, attention deficit hyperactivity disorder, personality disorders, autism, post-traumatic stress disorder, anxiety disorders, panic disorder, depression, chronic stress-related depression, delusional disorder, obsessive-compulsive disorder, anorexia nervosa, bulimia nervosa, obesity, cerebral white matter lesions, neurodegenerative diseases, diabetic neuropathy, traumatic nerve injury, neurodegenerative diseases, neuropathic pain, epilepsy (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 encephalitis), chronic traumatic encephalopathy,This includes, but is not limited to, CTE, myotonic dystrophy, multiple sclerosis, Schwannoma, neurofibromatosis, chronic inflammatory demyelinating polyneuropathy (CIDP), polyneuropathy, and schwannoma.

[0071] In the present invention, "prevention" refers to a Schwann cell precursor (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2 and combinations thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10 and combinations thereof, and CD56 + CD16 + This means any act of suppressing or delaying the onset of neurological diseases using a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising induced natural killer (drNK) cells expressing at least one selected from the group consisting of combinations thereof.

[0072] In the present invention, "treatment" refers to a Schwann cell precursor (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2 and combinations thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10 and combinations thereof, and CD56 + CD16 + This means any act of curing or improving the symptoms of a neurological disorder using a pharmaceutical composition for the prevention or treatment of a neurological disorder, comprising induced natural killer (drNK) cells expressing at least one selected from the group consisting of combinations thereof.

[0073] The "pharmaceutical composition" in the present invention may contain a pharmaceutically acceptable carrier and is formulated by conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injection solutions.

[0074] The pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention also includes fillers, bulking agents, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

[0075] Examples of oral solid dosage forms formulated from the pharmaceutical composition of the present invention include tablets, pills, powders, granules, and capsules. These solid dosage forms contain at least one excipient, such as starch, calcium carbonate, sucrose, or lactose, or gelatin, and may also contain lubricants such as magnesium stearate or talc, but are not limited to these.

[0076] Oral liquid formulations derived from the pharmaceutical composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and include, but are not limited to, water, diluents such as liquid paraffin, humectants, sweeteners, fragrances, and preservatives.

[0077] Examples of parenteral formulations derived from the pharmaceutical composition of the present invention include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. 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, cocoa butter, lauric butter, and glycerol gelatin.

[0078] In the present invention, the dosage of the pharmaceutical composition will vary depending on the patient's condition, weight, age, severity of the disease, form of the drug, route of administration, and duration, but will be appropriately selected by those skilled in the art.

[0079] The pharmaceutical compositions of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes, such as orally, or by intraperitoneal, intravenous, intramuscular, subcutaneous, intrauterine, dura mater, or intraventricular injection.

[0080] In one specific example of the present invention, when neurite outgrowth induced by SCP / SCP-SC and NK cells of the present invention was confirmed, superior neurite outgrowth was observed in the SCP-SC-only treatment group and the SCP-SC-and-drNK combined treatment group compared to the control group. In particular, the combined SCP-SC-and-drNK treatment group showed a significantly higher effect than the SCP-SC-only treatment group. Furthermore, in the case of a partial nerve injury model, the nerve outgrowth effect was higher with SCP compared to NCSC, and the nerve outgrowth effect was significantly higher in the combined SCP-SC-and-drNK treatment group compared to the SCP-only treatment group.

[0081] In one specific example of the present invention, when the composition of the present invention was transplanted into a sciatic nerve injury model mouse, the average motor function recovered better in the group that received a combination of SCP and drNK compared to the group treated with SCP alone, and it was confirmed that this group exhibited superior regenerative therapeutic effects.

[0082] This suggests that treating nerve injury disease models with induced Schwann cell precursors (SCPs) produced by the pluripotent stem cells (PSCs) of the present invention, and Schwann cells (SCs) differentiated therefrom, either alone or in combination with natural killer cells, exhibits superior regenerative and therapeutic effects on the damaged nervous system.

[0083] To achieve the above objective, yet another aspect of the present invention is a Schwann cell precursor (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2 and combinations thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10 and combinations thereof, and CD56 + CD16 + The present invention provides a cell therapy agent for the prevention or treatment of neurological diseases, comprising induced natural killer (drNK) cells expressing at least one selected from the group consisting of combinations thereof.

[0084] The terminology used here is as previously described.

[0085] In this invention, "cell therapy agent" means a pharmaceutical product (as defined by the U.S. FDA) that is produced by separating, culturing, and performing special operations on cells and tissues from an individual, and is used for therapeutic, diagnostic, and preventive purposes. It also means a pharmaceutical product used for therapeutic, diagnostic, and preventive purposes through a series of actions, such as growing and selecting living self, allogeneic, or heterogeneic cells outside the body, or altering the biological properties of cells by other means, in order to restore the function of cells or tissues.

[0086] The cell therapy composition may exert preventive or therapeutic efficacy against neurological diseases by containing Schwann cell precursors or Schwann cells differentiated therefrom, produced by the method of the present invention, and induced natural killer cells.

[0087] The cell therapy composition contains 1.0 × 10 to 1.0 × 10 units of Schwann cell precursors, Schwann cells, and induced natural killer cells relative to the total weight of the composition. 10Individual cells / ml, specifically 1.0 × 10⁶ 6 ~1.0×10 9 This includes, but is not limited to, individual cells / ml.

[0088] The cell therapy composition can be administered in the form of a unit-dose pharmaceutical preparation suitable for administration to a patient by conventional methods in the pharmaceutical field, and the preparation can be administered in an effective dose by administering it in one or several divided doses. Suitable dosage forms for the above purpose include parenteral administration preparations such as injection ampoules, infusion bags, and aerosol preparations. The injection ampoules can be mixed with an injection solution immediately before use, and the injection solution can be physiological saline, glucose, mannitol, Ringer's solution, etc. Infusion bags can be made of polyvinyl chloride or polyethylene, and examples include infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo.

[0089] In addition to the active ingredient, the aforementioned pharmaceutical preparation may further contain one or more pharmaceutically acceptable common inert carriers, such as preservatives, analgesics, solubilizers, and stabilizers in the case of injection preparations, and a base, excipients, lubricants, and preservatives in the case of topical administration preparations.

[0090] The cell therapy composition or pharmaceutical preparation thereof manufactured in this manner may be administered together with or in the form of a mixture of other cells used for the treatment of neurological diseases, using administration methods commonly used in the art. Specifically, it may be directly engrafted or transplanted to the diseased site of a patient requiring treatment, or directly transplanted or injected into the abdominal cavity, but is not limited to these methods. Furthermore, all administration methods are possible, including non-surgical administration using a catheter, and surgical administration methods such as injection and transplantation after incision of the diseased site. In addition, in addition to parenteral administration by conventional methods, such as direct administration to the lesion, transplantation by intravascular injection is also possible.

[0091] The cell therapy composition may be administered at a dose of 0.0001 to 1,000 mg / kg per day, specifically 0.01 to 100 mg / kg, and the administration may be once a day or divided into several doses. However, it should be understood that the actual dose of the active ingredient must be determined by considering various relevant factors such as the disease being treated, the severity of the disease, the route of administration, the patient's weight, age, and sex. Therefore, the aforementioned dose does not limit the present invention in any case.

[0092] To achieve the above objective, yet another aspect of the present invention provides a method for preventing or treating a neurological disease, comprising the step of administering a pharmaceutical composition for the prevention or treatment of a neurological disease, comprising the step of administering to a non-human individual suspected of having a neurological disease, the composition comprising Schwann cell precursors (SCPs) or Schwann cells differentiated therefrom (SCs) and natural killer (NK) cells as active ingredients.

[0093] Specifically, a Schwann cell precursor (SCP) expressing at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof, or a Schwann cell (SC) expressing at least one selected from the group consisting of S100B, SOX10, and combinations thereof, and CD56 + CD16 +The present invention provides a method for preventing or treating neurological diseases, comprising the step of administering a pharmaceutical composition for the prevention or treatment of neurological diseases to an individual suspected of having a neurological disease other than a human, comprising natural killer (NK) cells expressing at least one selected from the group consisting of combinations thereof.

[0094] The terminology used here is as previously described.

[0095] The term "administration" means introducing the composition of the present invention into an individual by any appropriate method, and the administration route of the composition can be any common route that can deliver it to the target tissue. This includes, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, and intranasal administration.

[0096] The term "individual" as used herein means any animal other than humans that has developed or is at risk of developing a neurological disease, including monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs. Any type of individual is acceptable as long as the pharmaceutical composition of the present invention can be administered to the individual to effectively prevent or treat the disease. [Examples]

[0097] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention, and the present invention is not limited to these examples. [Examples]

[0098] Differentiation of Schwann cell precursors (SCPs) from human pluripotent stem cells (PSCs) We devised a novel method that improves upon the conventional production method (protocol 1, A in Figure 1) for differentiating pluripotent stem cells into Schwann cell precursors. To confirm the effects of FGF2, LDN193189, all-trans-retinoic acid (RA), StemRegenine1 (SR1), and Dorsomrphin as novel candidate factors for promoting Schwann cell precursor differentiation, we compared and analyzed the characteristics of cells differentiated using eight methods (Protocols 1-8), as shown in Figures A and B.

[0099] First, human pluripotent stem cells (PSCs), such as human induced pluripotent stem cells (induced pluripotent stem cells) derived from human neonatal foreskin fibroblasts (Newborn foreskin fibroblasts, catalog number CRL-2097; ATCC) and human embryonic stem cells (H9 ESC, WiCell), were cultured as follows. For feeder-free culture, the cells were grown in mTeSR1 medium (StemCell Technologies) on dishes coated with growth factor-reduced Matrigel (BD biosciences), with the medium changed daily. To obtain Schwann cell precursors derived from human PSCs, colonized PSCs were again plated onto culture dishes coated with growth factor-reduced Matrigel. The following day, the culture medium was neutralized by replacing the human PSC culture medium with modified neural differentiation medium (NDM) containing SB431542 and CT99021, and the cells were cultured for 6 days to form neural rosettes. Specifically, the NDM contained either advanced DMEM / F12 and neurobasal medium (1:1 mixture) containing 1x N2, 1x B27, 0.005% BSA, 2mM Glutamax, 0.11mM β-mercaptoethanol, 3μM CT 99021 (Tocris Biosciences), and 20μM SB431542 (Tocris Biosciences), or, as shown in Figures 1A and B, the NDM medium further contained 20μg / ml FGF2 (Peprotech) or 100nM LDN193189 (Medchemexpress). Six days after differentiation, the NDM medium was replaced with a 50 ng / ml NRG1-containing neuronal induction medium [Schwann cell precursor induction medium (SCPDM)].As shown in Figure 1B, the SCPDM medium contains 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. The SCPDM was replaced every two days, and when the cells reached 80% density (Confluence), they were treated with Accutase to isolate and cultured for an additional six days. The cells were further cultured in SCPDM to increase their volume. Approximately 18 days after differentiation, to confirm whether or not SCP was produced, the expression of the SCP marker gene SOX10 was first checked by qPCR, and high expression was confirmed in Protocol 1, Protocol 4, Protocol 5, and Protocol 7 (Figure 1C). Furthermore, when the expression levels of CD49d, ERBB3, and PLP1, known as SCP-labeled genes, were confirmed by qPCR, it was found that they were expressed more highly in the novel differentiation methods, Protocol 4 and Protocol 5, compared to the conventional differentiation method for Protocol 1 (Figure 1, D). In particular, for ERBB3, expression levels were 2.15 times higher than the conventional method in Protocol 1 (average 2.15 times higher than GAPDH), 5.99 times higher in Protocol 4 (average 5.81 times higher than GAPDH), and 2.7 times higher than the conventional method in Protocol 5. Similarly, for PLP1, expression levels were 7.39 times higher in Protocol 1 (average 7.2 times higher than GAPDH), 17.2 times higher in Protocol 4 (average 18.9 times higher than GAPDH), and 2.3 times higher than the conventional method in Protocol 5. Protein expression of SOX10, GAP43, and IGFBP2 were confirmed as Schwann cell precursor labeling factor proteins by immunohistochemical staining in Protocols 1, 4, and 5 (Figure 1E). SCPDM was used for the induction and maintenance of PSC-SCP. [Examples]

[0100] Differentiation and production of Schwann cells (SCs) from SCPs To confirm the Schwann cell differentiation ability of SCPs differentiated by the novel protocol, SC differentiation was performed along with conventional iSCPs using a conventionally known SCP-SC differentiation method. To differentiate SCPs into Schwann cells, the aforementioned SCPs and SCPs produced by the somatic cell reprogramming method (iSCPs) were cultured on plates coated with Matrigel in Schwann cell differentiation medium (SCDM). The SCDM contained 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. Three days after culturing, the culture medium was replaced with SCDM containing 1% FBS, 200 ng / ml NRG1, and 10 ng / ml PDGF-BB (Thermo Fisher Scientific), but without forskolin or retinoic acid. Two days later, the culture medium was replaced with SCDM (Schwann cell medium, SCM) containing 1% FBS and 200 ng / ml NRG1, but without forskolin, retinoic acid, or PDGF-BB. The cultured cells were maintained in SCM for expansion. Schwann cells were generated 2-3 days after culturing in SCM. On day 7 after differentiation, qPCR was used to analyze the expression levels of S100b, NGFR, MPZ, and EGR2 as SC-labeling genes. No significant difference was observed compared to iSCP-SC, which has been conventionally confirmed to have Schwann cell function (Figure 2A). Immunocytochemical analysis confirmed that most of the differentiated SCs were positive for S100B and SOX10, which are SC lineage-specific proteins (Figure 2B). Therefore, it was confirmed that Schwann cell precursors differentiated by the novel method can successfully produce Schwann cells in a short time (approximately 7 days), comparable to cells differentiated by the conventional method. [Examples]

[0101] Confirmation of higher neurotrophic factor GDNF and IGFBP-2 mRNA expression and protein secretion characteristics in induced SCPs compared to conventional Schwann cell precursors (NCSCs). To confirm the difference between the induced SCP of the present invention and NCSC, a representative Schwann cell precursor known conventionally, in terms of the protein secretion capacity of cells, NCSC were first differentiated from human PSCs (H9 ESCs) using the following method.

[0102] Specifically, the isolated PSCs were plated onto a culture dish coated with Matrigel, and the following day, 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 8 ng / ml The culture medium was replaced with NCSC induction medium (NCSCIM) containing FGF2 (Peprotech). The culture medium was changed daily. NCSCs were produced approximately 20 days after growth on NCSCIM. Unless otherwise specified, all reagents were purchased from Thermo Fisher Scientific.

[0103] Quantitative analysis of the neurotrophic factors GDNF and IGFBP-2 gene expression levels in hPSCs, differentiated NCSCs (as described above), and induced SCP cells of the present invention was performed by qPCR. The results showed that these levels were significantly higher in SCP compared to NCSC (Figure 3A). To obtain a conditioned medium (CM) for comparative analysis of proteins secreted from NCSC and induced SCP that affect nerve regeneration and growth, 10 5 Individual SCP and NCSC cells were seeded into 30 mm culture dishes with 2 ml of culture medium. After 48 hours, the culture medium was filtered through a 0.22 μm filter (Millipore) as described above. To measure the concentration of secreted neurotrophic factor GDNF, ELISA was performed on the SCP and NCSC-derived conditional culture media according to the manufacturer's protocol (Abcam).

[0104] As a result, it was confirmed that SCP (16.6 pg / ml) secreted more than NCSC (3.3 pg / ml) (left side of B in Figure 3). To measure cytokine levels in NCSC-CM and SCP-CM, a proteome profiler array (Proteome Profiler Human XL Cytokine Array Kit, ARY022B; R&D system) was used according to the manufacturer's guidelines. In this example, images were obtained using an Amersham imager 600 (GE Healthcare Life Sciences), and then quantitatively analyzed using ImageJ (Open source software). It was confirmed that SCP (1150.5 MPD) secreted more than NCSC [157 MPD (Mean Pixel Density)] (right side of B in Figure 3). Therefore, it is inferred that, compared to NCSC, the higher secretion of nerve growth factor in SCP of the present invention will result in a greater effect on nerve growth and regeneration. [Examples]

[0105] Analysis of the phenotypic characteristics of induced natural killer (drNK) cells To obtain induced drNK cells, PBMCs isolated from human peripheral blood using a Ficoll concentration gradient were transformed with the reprogramming factor OSKM. Subsequently, the PBMC cells and polyblen (4 μg / ml) were cultured together for 1 day, and the following day, 3 × 10⁶ cells were cultured in a 48-well culture dish. 5 The transformed cells were cultured for a further 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, and 20 ng / ml Human TPO). The cells were then cultured for 18 to 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, and 2 μM StemRegenin I).

[0106] To confirm whether drNK cells were generated by the direct reprogramming described above, the cells were stained with anti-CD56-APC (Biolegend) antibody and anti-CD16-PE (Biolegend) antibody, which are markers for NK cells, and then flow cytometry was used to identify the NK cells (CD56 + and CD16 + The ) groups were analyzed. As control cells, fresh primary NK (pNK) cells isolated from PBMCs, or pNK (ApNK) cells activated with 200 IU / ml Human IL-2 and 20 ng / ml Human IL-15 for 4 to 14 days, and NK cell lines (NK92, ATCC) were used.

[0107] In the major cell populations, drNK:CD56 was expressed according to the expression levels of the CD56 and CD16 markers, respectively.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 can be divided into (Figure 4A and B). Here, the most commonly used pNK (CD56) dim Depending on the intensity of the CD56 and CD16 fluorescence of pNK, dim(10 4 below), bright(10 4 ~10 5 ), superbright(10 5 (The above) was written. In particular, when comparing NK cell receptor expression, CD56 dim Compared to pNK cells, drNK cells showed higher expression of NK cell activating receptors such as CD69, NKG2D, DNAM-1, and NKp46 (Figure 4C). [Examples]

[0108] Quantitative analysis of drNK expression cytokine / chemokine genes. To produce iPSC-NK cells as a control group for comparison with drNK, dedifferentiated stem cells (iPSCs) were treated with ReLeSR (Stem Cell Technologies) to dissociate into single cells, and 1x penicillin / streptomycin (Invitrogen), 40 ng / ml SCF (Invitrogen), 20 ng / ml VEGF (R & D), and 20 ng / ml BMP4 (R & D) were added to STEMdiff APEL2 medium (Stem Cell Technologies), which is the culture medium composition for spin embryoid bodies, and 3 × 10⁶ cells were added. 4Cells suspended in a cell / ml concentration were seeded at a rate of 3,000 cells per well in a round-bottom 96-well plate. After centrifugation at 8°C and 1,500 rpm for 4 minutes, the cells were cultured in a 37°C incubator for 3-4 days, and then half of the culture medium was replaced with fresh medium and cultured for 9-11 days. On days 9-11 after spin embryoid differentiation, spin embryoids from 6-8 wells of a 96-well plate were transferred to 1 well of a 24-well plate coated with 2% gelatin. The differentiation culture medium consisted of 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), and 10 ng / ml Cells were cultured with the cytokine Flt3L (Peprotech), and then cultured for 28 days in NK differentiation medium to which cytokines other than IL-3 were further added every 5-7 days. These 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.

[0109] To quantitatively analyze the gene expression of cytokines / chemokines expressed in the aforementioned drNK cells, along with that of control NK cells (NK-92 and iPS-NK cells), qRT-PCR was performed.

[0110] As a result, it was confirmed that the expression of drNK was relatively increased compared to the NK-92 and iPS-NK groups. If we set the expression level in NK-92 as 1 and express the expression levels in iPS-NK and drNK as multiples, then the following ratios were obtained: [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)] (Figure 5). Therefore, a comparative analysis of cytokine / chemokine molecular target gene expression levels that play an important role in NK function in NK-92, iPS-NK, and drNK revealed that 10 genes (CCL5, IFN-γ, CXCL11, CXCL12, GDNF, VEGF, XCL1, IL16, LIF, LTB) are overexpressed in drNK. [Examples]

[0111] Identification of drNK secretion factors using human proteomic cytokine arrays The secretory capacity of cytoplasmic substances, particularly cytokines, that affect nerve regeneration in drNK cells was analyzed in culture medium. Specifically, to obtain a conditioned medium (CM) for NK cells, drNK cells and CD56 dim Place pNK cells in a culture dish. 6 The cells were cultured at a density of cells / ml. After 24 hours, the culture medium was filtered through a 0.22 μm filter (Millipore) as described above. drNK-CM and CD56 dimTo measure cytokine levels in pNK-CM, a proteome profiler array (Proteome Profiler Human XL Cytokine Array Kit, ARY022B; R&D system) was used according to the manufacturer's guidelines. ImageJ software was used for quantitative analysis of the final images.

[0112] The results, according to ImageJ (image intensity = 1,000 Mean Pixel Density (MPD) or higher), showed 56 types of drNK secreted tanpaquinones (RANTES (mean = 81,174 MPD), DPPIV (mean = 78,046 MPD), CD31 (mean = 33,901 MPD), TIM-3 (mean = 31,688 MPD), Emmprin (mean = 29,204 MPD), MIP-1α / MIP-1β (mean = 27,937 MPD), GM-CSF (mean = 22,299 MPD), Fas Ligand (mean = 21,602 MPD), MIF (mean = 19,398 MPD), IL-16 (mean = 16,867 MPD), IL-17A (mean = 8,249 MPD), Flt-3 Ligand (mean = 5834 MPD), ICAM-1 (mean = 4,532 MPD), M-CSF (mean = 2,867 MPD), FGF-19 (mean = 2481 MPD), Serpin E1, MIP-3β (mean = 2,165 MPD), IL-18 (mean = 2,144 MPD), IL-32 (mean = 1,950 MPD), Angiogenin (mean = 1,901 MPD), IL-1α (mean = 1,895 MPD), Cystatin C (mean = 1,792 MPD), Resistin (mean = 1,699 MPD), GDF-15 (mean = 1,635 MPD), Angiopoietin-2 (mean = 1,558 MPD), TNF-α (mean = 1,546 MPD), PDGF-AA (mean = 1,536 MPD), Apolipoprotein AI (mean = 1,507 MPD), Osteopontin (mean = 1,489 MPD), Dkk-1 (mean = 1,447 MPD), uPAR (mean = 1,422 MPD), Endoglin (mean = 1,411 MPD), IFN-γ (mean = 1,394 MPD), FGF basic (mean = 1,381 MPD), SDF-1α (CXCL12) (mean = 1,378 MPD), IL-1β (mean = 1,363 MPD), RAGE (mean = 1,327 MPD), EGF (mean = 1,277 MPD), IL-8 (mean = 1,252 MPD), IL-27 (mean = 1,208 MPD), BAFF (mean = 1,191 MPD), CD40 ligand (mean = 1,159 MPD), IL-34 (mean = 1,129 MPD), VCAM-1 (mean = 1,127 MPD)IL-1ra (mean = 1,113 MPD), MCP-1 (mean = 1,094 MPD), Kallikrein 3 (mean = 1,088 MPD), IL-12 p70 (mean = 1,088 MPD), IL-11 (mean = 1,087 MPD), ST2 (mean = 1,059 MPD), MMP-9 (mean = 1,058 MPD), IL-22 (mean = 1,057 MPD), C-Reactive Protein (mean = 1,041 MPD), BDNF (mean = 1,034 MPD), Vitamin D BP (mean = 1,032 MPD), and Lipocalin-2 (mean = 1,004 MPD) were identified (Figure 6A). Control group CD56, dim 28 proteins secreted at quantitatively higher levels compared to pNK: [IL-16 (5.58x), BAFF (5.23x), CD31 (4.36x), ICAM-1 (3.57x), Emmprin (3.39x), VCAM-1 (3.33x), Flt-3 Ligand (3.32x), Cystatin C (3.16x), C-Reactive Protein (2.58x), IL-27 (2.44x), TNF-α (2.22x), IL-32 (2.16x), TIM-3 (2.01x), IL-12 p70 (1.94x), uPAR (1.53x), IL-18 We identified Bpa (1.40x), MIF (1.39x), Dkk-1 (1.35x), IL-11 (1.33x), GM-CSF (1.30x), RANTES (1.29x), Endoglin (1.24x), IL-22 (1.16x), RAGE, Osteopontin (1.06x), GDF-15 (1.02x), Kallikrein 3 (1.02x), and Angiopoietin-2 (1.02x) (Figure 6B). We identified three proteins specifically identified in drNK (DPP4, M-CSF, BDNF) (Figure 6C). [Examples]

[0113] Confirmation of drNK's nerve clearing effect and verification of its CD16 expression dependency. To confirm the selective removal effect of damaged neurons by NK cells, a partially damaged SH-SY5Y model was used. To induce damaged neurons, SH-SY5Y cells were treated with 200 μM H2O2 and then treated in DMEM / F12 (1:1) medium containing 10% FBS for 24 hours. It was confirmed that most of the cells were labeled with ROS (MitoSox Red, Thermofisher Scientific), indicating damaged cells (Figure 7A). To induce partially damaged neurons, SH-SY5Y cells were treated with 200 μM H2O2 and then treated in DMEM / F12 (1:1) medium containing 10% FBS for 24 hours. Cells that were not treated with H2O2 and treated SH-SY5Y cells were mixed in a 1:3 ratio and plated. The following day, CD56 dim When co-cultured with pNK and drNK (E:T=0.25:1) and the number of ROS-positive neurons was checked after 24 hours, the ratio of ROS-positive neurons was higher in CD56 cells compared to the control group not co-cultured with NK cells (56.5%). dim Not only was a significant decrease observed in pNK (22.0%) and drNK (8.9%), but CD56 dim It was confirmed that drNK removed more than twice as many damaged nerve cells compared to pNK (Figure 7B).

[0114] To confirm the correlation between the debridement effect of natural killer cells and CD16 expression levels, SH-SY5Y neurons, a model of damaged nerve from the above example, were treated with H2O2 to induce nerve damage. These cells were then co-cultured with CD16 antibody and NK cells (E:T=1:1), and the debridement effect of NK cells was analyzed after 4 hours. Specifically, SH-SY5Y cells were treated with 200 nMH2O2 for 24 hours in DMEM / F12 (1:1) medium containing 10% FBS to induce nerve cell damage. Cell tracker-labeled NK cells, damaged neurons, and CD16 antibody (Biolegend, 1:20 concentration) were cultured individually or co-cultured (E:T=1:1), and after 4 hours, they were examined by Propidium iodide (PI, Thermofisher Scientific) staining and flow cytometry (Figure 7C).

[0115] As a result, using the effect of removing damaged nerve cells when treated alone without antibodies as the baseline (100%), the effect of removing each type of damaged nerve cell with CD16 antibody was compared to that with CD56 antibody. dim pNK (58.1%), CD56 bright A decrease in pNK (50.1%), NK92 (82.6%), and drNK (30.9%) was confirmed. In conclusion, it was confirmed that the inhibitory effect of the CD16 antibody is proportional to the expression level of CD16, and in the drNK cells of the present invention, the inhibitory effect of the CD16 antibody was 69.1%, confirming that the maximum inhibitory effect was obtained (Figure 7D). A list of antibodies used in the present invention is shown in Table 1.

[0116] [Table 1] [Examples]

[0117] Confirmation of neurite outgrowth caused by SCP / SCP-SC and NK cells. To confirm neurite outgrowth by SCP / SCP-SCs and NK cells, a scratch model of neurons differentiated from hiPSCs and a partially damaged SH-SY5Y model were used (Figure 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, and 0.1 mM β-mercaptoethanol) in a 500 × 500 μm colony size for 7 days in suspension. Subsequently, they were subjected to rotary orbital culture at a speed of 20-22 rpm in neurosphere (NS) culture medium (DMEM / F12 and 1X N2, 1X B27, 1% Penicillin / Streptomycin, 20 ng / ml hEGF, 20 ng / ml bFGF, and 10 ng / ml hLIF). NS cells were subcultured to a size of 250 × 250 μm every 5-7 days. During the second subculture (NS p2), NS p2 cells were attached to a coverslip coated with 0.01% PLL (Poly-L-lysine) at 4°C for 24 hours. These were cultured for 7-14 days in 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). Damage experiments were performed after neurite extension from the NS cells. Neurite damage from the NS body was achieved by scratching (damaging) the tip of a sterile pipette tip. The following day, SC (10,000 cells / cm²) was measured. 2) and drNK (10,000 cells / ml) were cultured together. Cell morphology and βIII tubulin antibody (Cat. no. ab78078, abcam, 1:200 dilution) were confirmed by immunohistochemical staining after 24 hours of co-culture. Images before and after injury, and immunostaining of SCP-SC (GFP labeled) and drNK after 24 hours of co-culture showed neurite (TUJ1: red) and cell nuclei (DAPI: dark blue), allowing us to confirm the difference in neurite elongation in each experimental group (Figure 8B).

[0118] In the above examples, SCP-SC alone and SCP-SC + drNK combined treatments were applied according to the method shown in Figure 8B, and neurite elongation after 48 hours of culture was quantitatively analyzed. As a result, the SCP-SC alone treatment group (average length = 251.7 μm) and the SCP-SC and drNK combined treatment group (average length = 315.9 μm) showed superior neurite elongation compared to the control group (average length = 157.7 μm) (Figure 8C). In particular, the SCP-SC and drNK combined treatment group showed an average elongation that was more than 1.25 times higher than the SCP-SC alone treatment group.

[0119] To confirm neurite outgrowth by SCP / SCP-SC and NK cells in a partial nerve injury model, SH-SY5Y cells were treated with 200 μM H2O2 as in Example 7B, and then treated in DMEM / F12 (1:1) medium containing 10% FBS for 24 hours. Untreated cells and treated SH-SY5Y cells were mixed in a 1:3 ratio, and 2 × 10⁶ cells were placed on a Matrigel-coated plate in differentiation medium (DMEM / F12 (1:1) medium containing 1 μM Retinoic acid and 2% FBS). 3 cells / cm 2They were seeded and 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) as described above [ratio = 2 (neurons): 1], and cultured for another 2 days before analyzing neurite outgrowth. Images were obtained using an Axio Vert.A1 microscope (Carl Zeiss). When measuring the length of neurites in each image using AxioVs40 v4.8.2.0 software, compared to the control group (average length = 43.5 μm), neurites were confirmed in the SCP-SC treatment group (average length = 72.9 μm), CD56 dim pNK treatment group (average length = 57.4 μm), drNK treatment group (average length = 59.8 μm), SCP-SC and CD56 dim pNK treatment group (average length = 87.3 μm), SCP-SC and drNK treatment group (average length = 158.6 μm), NGF treatment group (average length = 60.1 μm), and NGF and drNK treatment group (average length = 67.2 μm). Growth promotion was confirmed in the pSC treatment group (average length = 45.8 μm), pSC and CD56 dim pNK treatment group (average length = 57.1 μm), pSC and drNK treatment group (average length = 65.1 μm) (D in Figure 8). Here, compared to pSC, in SCP-SC it was more than 1.59 times on average, CD56 dim compared to pNK, in drNK it was 1.04 times on average, and compared to the SCP-SC single treatment group, in the combined treatment group of SCP-SC and drNK it was more than 2.17 times on average, and it was confirmed that the neurite elongation effect was much higher.

[0120] In the combined treatment group of SCP and NCSC, in the SCP treatment group (average length = 71.1 μm), SCP and drNK treatment group (average length = 81.7 μm), SCP and CD56 dim pNK treatment group (average length = 53.7 μm), NCSC treatment group (average length = 53.7 μm), NCSC and drNK treatment group (average length = 67.3 μm), NCSC and CD56 dimIt was confirmed that it became the pNK treatment group (average length = 62.5 μm) (E in FIG. 8). Therefore, compared with NCSC, in SCP, it was 1.32 times or more on average, and compared with the SCP single treatment group, in the combined treatment group of SCP and drNK, it was 1.14 times or more on average, and it was confirmed that the nerve elongation effect was much higher.

Example

[0121] Promotion of nerve regeneration and therapeutic effect by single or combined transplantation of SCP and NK cells in a sciatic nerve injury animal model To analyze the nerve recovery and therapeutic effects of Schwann cell precursors and natural killer cells in vivo by single or combined transplantation, a sciatic nerve injury model mouse was used (A in FIG. 9). First, the central region of the left sciatic nerve of 8-week-old C57BL / 6 male mice was transected to cause injury, and a nerve defect of 2 - 3 mm was formed. The Schwann cell precursor cells differentiated in Example 1 and NCSC differentiated in Example 3 were diluted with Matrigel (2×10 4 cells / μl), and 1×10 5 cells (5 μl of cell suspension containing SCP labeled with GFP by lentivirus infection) were transplanted into the nerve defect site. At the same time, the induced natural killer (drNK) cells (1×10 7 cells / 150 μl) were transplanted into the tail vein (A in FIG. 9). Sixteen weeks after transplantation, the sciatic nerves of each group were sampled, and immunohistochemical analysis was used to confirm the promotion of nerve regeneration and therapeutic effects by single transplantation of NCSC cells and combined transplantation with SCP.

[0122] As a result, immunohistochemical analysis confirmed that the regeneration of MBP-positive nerve cells was superior in the group transplanted with SCP compared with the group transplanted with NCSC. Furthermore, when the ratio of MBP-positive nerve cells was measured by quantitative analysis, it was confirmed that it was about 2.1 times higher in the group transplanted with SCP [control group NCSC (80.6 cells / 0.2 mm 2 ), SCP of the present invention (195.3 cells / 0.2 mm 2 )] (C in FIG. 9).

[0123] In the aforementioned sciatic nerve injury model, a Rotarod test was performed 8 weeks after NCSC transplantation, SCP transplantation, and combined SCP and NK cell transplantation to verify the effect on motor function. As a result, it was confirmed that motor function improved by approximately 1.45 times or more in the group transplanted with the SCP of the present invention compared to the control group with NCSC [control group NCSC (55.6 sec), SCP of the present invention (80.94 sec)] (Figure 9D).

[0124] Similarly, an analysis of the average motor function recovery in the combined SCP and drNK transplantation group compared to the SCP-only treatment group using the Rotarod test revealed that the combined SCP and drNK transplantation resulted in a therapeutic effect approximately 1.26 times better [control group: SCP alone (100 sec), drNK (89.0 sec), SCP + drNK (126.6 sec)] (Figure 9E). [Examples]

[0125] Nerve regeneration promotion and therapeutic effects of SCP-SC and NK cell transplantation alone or in combination. To analyze nerve recovery and therapeutic effects of transplantation of nerve growth factor (NGF), Schwann cells, and natural killer cells, either individually or in combination, a sciatic nerve partial injury model was used. First, partial sciatic nerve injury was induced in 8-week-old immunodeficiency Balb / c-nude male mice by pinching the central region of the sciatic nerve with forceps. At the injury site, PBS containing 20 μg / ml NGF (Peprotech) or SCP-SCs cells were placed in Matrigel (2 × 10⁻¹⁴⁻¹ 4 5 μl of each cell suspension, prepared by diluting (cells / μl), was transplanted into the nerve defect site. At the same time, as shown in Figure 10A, each experimental group was given CD56 bright pNK or drNK cells (1 × 10⁻¹⁰) 7 Cells were transplanted into the tail vein. Four weeks after transplantation, the sciatic nerves of each group were sampled, and the efficacy in treating peripheral nerve diseases was confirmed by morphological analysis using hematoxylin and eosin staining (H&E). Figure 10A shows each group (NGF, CD56 bright pNK, drNK, SCP-SC, CD56bright This figure shows representative staining results for pNK+SCP-SC and drNK+SCP-SC. Morphologically, in the single-treatment group, conventional CD56 dim The present invention's drNK was shown to be superior to pNK in terms of nerve recovery and regeneration effects. Furthermore, it was confirmed that the SCP-SC+NK combined treatment group showed even greater relative regenerative therapeutic effects compared to the group treated with drNK alone (Figure 10A).

[0126] In sample A of Figure 10, immunohistochemical analysis confirmed the presence of TUJ1-positive cells, which are more positive for SCP-SC and CD56 compared to the conventional control drug NGF. bright In the groups treated with pNK or drNK alone, a significantly higher nerve regeneration-promoting effect was confirmed [NGF (8.0%), SCP-SC (28.6%), CD56]. bright pNK (16.3%), drNK (25%). Here, conventional CD56 bright Compared to pNK, the drNK of the present invention was confirmed to have more than 1.53 times superior nerve recovery and regeneration effects. Compared to single transplantation, the SCP-SC + drNK combined treatment group was confirmed to have the most superior regenerative therapeutic effect. In particular, CD56 bright Compared to the pNK+SCP-SC combined treatment group, the drNK+SCP-SC combined treatment group showed an average nerve regeneration effect that was more than 1.98 times higher [SCP-SC+drNK(47.6%),CD56]. bright pNK+SCP-SC (24.0%) (Figures 10B and C).

[0127] Using the sciatic nerve model confirmed in Figure 10 of the above example, immunohistochemical analysis and qRT-PCR analysis of the Schwann cell marker S100 and the myelination marker MBP were performed four weeks post-transplant to analyze the transplantation status and degree of myelination in the drNK, SCP-SC, and drNK+SCP-SC groups. As a result, it was confirmed that the protein and gene expression of S100 (Figure 11 A and B) and MBP (Figure 11 C and D) were significantly increased relative to the single-transplant group in the regenerated area of ​​the drNK+SCP-SC combined transplanted group [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)] compared to the single-transplant group. In particular, when comparing myelin-positive gene expression levels, it was confirmed that the combined treatment group showed expression levels approximately 1.37 times higher than the SCP-SC mono-treatment group.

[0128] To confirm the recovery of motor function through nerve regeneration after sciatic nerve injury as shown in Figure 10 of the above example, a Rotarod test was performed 4 weeks after transplantation. In the single-transplant group, the SCP-SC transplant group showed the best motor function recovery effect (average NGF of 1.25 times or more), and CD56 bright Compared to pNK, the drNK transplant group showed superior motor function recovery [NGF(46sec),CD56 bright [pNK (42 sec), drNK (54 sec), SCP-SC (57.7 sec)]. Furthermore, among the experimental groups, the drNK + SCP-SC combined implantation group showed the greatest improvement in motor function, compared to the control group CD56. bright In the pNK+SCP-SC composite implantation group, a relatively low effect was observed [drNK+SCP-SC(68.4sec),CD56]. bright [pNK + SCP-SC (53.3 sec)]. In particular, the SCP-SC and drNK combined transplant group showed a statistically significant improvement in motor function, on average more than 1.18 times, compared to the SCP-SC transplant group, which showed the highest efficacy among the single transplant groups (Figure 12).

[0129] From the above description, those skilled in the art in the field to which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. The present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A pharmaceutical composition for the prevention or treatment of neurological diseases, comprising Schwann cell precursors (SCPs) or Schwann cells differentiated therefrom (SCs), and natural killer (NK) cells as active ingredients, The Schwann cell precursor (SCP) expresses at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof. The Schwann cells (SCs) express at least one selected from the group consisting of S100B, SOX10, and combinations thereof. The aforementioned natural killer (NK) cells are CD56 ＋ CD16 ＋ It expresses at least one selected from the group consisting of combinations thereof. Pharmaceutical compositions for the prevention or treatment of neurological diseases.

2. The Schwann cell precursor (SCP) is (a) A step of culturing pluripotent stem cells in a first medium containing SB431542 and CT99021, (b) The step of culturing the cells cultured in step (a) in a second medium to which NRG1 (Neuregulin-1) is further added, It was produced by a method for generating Schwann cell precursors from pluripotent stem cells. A pharmaceutical composition for the prevention or treatment of neurological diseases as described in claim 1.

3. The first medium in step (a) further contains FGF2, and the second medium in step (b) further contains stem regenin I. A pharmaceutical composition for the prevention or treatment of neurological diseases as described in claim 1.

4. The Schwann cells (SCs) are, (a) A step of culturing pluripotent stem cells in a first medium containing SB431542 and CT99021, (b) A step of culturing the cells cultured in step (a) in a second medium to which NRG1 (Neuregulin-1) is further added, (c) A step of recovering Schwann cell precursors from the culture medium, (d) The step of culturing the recovered Schwann cell precursor in a third medium containing FBS and NRG1, These cells were produced using a method that generates Schwann cells from pluripotent stem cells. A pharmaceutical composition for the prevention or treatment of neurological diseases as described in claim 1.

5. The third medium in step (d) further contains at least one selected from the group consisting of retinoic acid, forskolin, and PDGF-BB. A pharmaceutical composition for the prevention or treatment of neurological diseases as described in claim 4.

6. The aforementioned natural killer (NK) cells are CD56 ｄｉｍ CD56 ｂｒｉｇｈｔ CD56 ｓｕｐｅｒｂｒｉｇｈｔ CD16 ｄｉｍ CD16 ｂｒｉｇｈｔ CD16 ｓｕｐｅｒｂｒｉｇｈｔ It expresses at least one selected from the group consisting of combinations thereof. A pharmaceutical composition for the prevention or treatment of neurological diseases as described in claim 1.

7. The aforementioned natural killer (NK) cells are (a) The step of introducing a reprogramming factor into the isolated cells, (b) From the day following the introduction of the reprogramming factor, the cells from step (a) are cultured in a first medium containing cytokines, growth factors and a GSK3β (Glycogen synthase kinase 3β) inhibitor to improve the efficiency of direct reprogramming, and ii) the cells are cultured in a second medium containing cytokines, growth factors and an AHR (Aryl hydrocarbon receptor) inhibitor to promote the production of natural killer cells. By a method for producing induced natural killer cells (drNK) from isolated cells It was created. A pharmaceutical composition for the prevention or treatment of neurological diseases as described in claim 1.

8. The induced natural killer (drNK) cells 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 the control group. A pharmaceutical composition for the prevention or treatment of neurological diseases according to claim 7.

9. The induced natural killer (drNK) cells express at least one protein selected from the group consisting of DPP4, M-CSF, and BDNF. A pharmaceutical composition for the prevention or treatment of neurological diseases according to claim 6.

10. The aforementioned neurological disorders include brain tumors, cerebral infarction, hypertensive cerebral hemorrhage, cerebral contusion, arteriovenous malformations, brain abscesses, encephalitis, chickenpox, epilepsy, concussions, cerebral palsy, mild cognitive impairment, dementia, spinal cord tumors, spinal arteriovenous malformations, spinal cord infarction, pain, headaches, migraines, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Button's disease, Kearns-Sayre syndrome (KSS), chronic progressive extraocular muscle palsy (CPEO), Meras syndrome (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), and MERRF syndrome (Myoclonic epilepsy with ragged-red). Fibres, NARP syndrome (neurogenic weakness with ataxis and retinitis pigmentosa), Reye's syndrome, MIRAS syndrome (mitochondrial recessive ataxis) Syndrome, degenerative neurological disorders, schizophrenia, attention deficit hyperactivity disorder (ADHD), personality disorders, autism, post-traumatic stress disorder (PTSD), anxiety disorders, panic disorder, depression, chronic stress-related depression, delusional disorder, obsessive-compulsive disorder, anorexia nervosa, bulimia nervosa, obesity, cerebral white matter lesions, neurodegenerative diseases, diabetic neuropathy, traumatic nerve injury, neurodegenerative diseases, neuropathic pain, epilepsy (epilepsy), chronic neuropathic pain, Guillain-Barré syndrome, myasthenia gravis, Rett syndrome, central sleep apnea, peripheral neuropathy Neuropathy, Charcot-Marie-Tooth disease, Spinal muscular atrophy (SMA), Autoimmune encephalitis, Chronic traumatic encephalopathy,It is characterized by being at least one disease selected from the group consisting of CTE, myotonic dystrophy, multiple sclerosis, Schwannoma, neurofibromatosis, chronic inflammatory demyelinating polyneuropathy (CIDP), polyneuropathy, and schwannoma. A pharmaceutical composition for the prevention or treatment of neurological diseases according to any one of claims 1 to 8.

11. A cell therapy agent for the prevention or treatment of neurological diseases, comprising Schwann cell precursors (SCPs) or Schwann cells differentiated therefrom (SCs), and induced natural killer (drNK) cells as active ingredients, The Schwann cell precursor (SCP) expresses at least one selected from the group consisting of GAP43, SOX10, IGFBP2, and combinations thereof. The Schwann cells (SCs) express at least one selected from the group consisting of S100B, SOX10, and combinations thereof. The natural killer (NK) cells express at least one selected from the group consisting of CD56 ＋ , CD16 ＋ and combinations thereof. Cell therapy agents for the prevention or treatment of neurological diseases.

12. The aforementioned natural killer (NK) cells are CD56 ｄｉｍ CD56 ｂｒｉｇｈｔ CD56 ｓｕｐｅｒｂｒｉｇｈｔ CD16 ｄｉｍ CD16 ｂｒｉｇｈｔ CD16 ｓｕｐｅｒｂｒｉｇｈｔ It expresses at least one selected from the group consisting of combinations thereof. A cell therapy agent for the prevention or treatment of neurological diseases according to claim 11.

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