HK1160789A - Composition comprising mesenchymal stem cells or culture solution of mesenchymal stem cells for the prevention or treatment of neural diseases - Google Patents

Abstract

Provided are a pharmaceutical composition for prevention and treatment of a neural disease including at least one selected from the group consisting of mesenchymal stem cells (MSCs), a culture solution of the MSCs, activin A, PF4, decorin, galectin 3, GDF15, glypican 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, progranulin, IL-4, a factor inducing expression thereof, and any combination thereof, and a method therefor.

Description

Composition comprising mesenchymal stem cell or mesenchymal stem cell culture solution for preventing or treating neurological disease

Technical Field

The present invention relates to a composition for preventing or treating Alzheimer's disease associated with neurite damage, which comprises Mesenchymal Stem Cells (MSCs), a culture solution of MSCs, a protein contained in the culture solution of MSCs, or a stimulating factor of a signal transduction system inducing the expression of the protein.

The present invention relates to a composition for preventing or treating a disease associated with neurite damage, which comprises Mesenchymal Stem Cells (MSCs), a culture solution of MSCs, a protein contained in the culture solution of MSCs, or a signal transduction system stimulating factor inducing the expression of the protein.

Background

Alzheimer's disease is a brain disorder that destroys brain cells caused by the destructive accumulation of amyloid beta protein and typically develops with age, a serious disease that causes language impairment and impairment of cognitive function. The progression of alzheimer's disease is in several stages and gradually destroys memory, reasoning, judgment, language, and even the ability to do simple work. Ultimately, loss of emotional control can cause a decline in the quality of life of a person. At present, alzheimer's disease is not completely cured, but drugs are clinically available to alleviate symptoms. However, these drugs have limited effect on patients. Approximately half of alzheimer's patients are not cured by initial drug therapy. Even if the initial drug treatment was successful, the symptoms were only slightly alleviated. Therefore, there is a need to develop a novel therapeutic approach that satisfies the medical needs, and the development of a therapeutic approach for alzheimer's disease will have great economic and social effects. It is known that cerebral cortex and hippocampus are damaged and cannot be recovered with the progress of alzheimer's disease, and thus, there is no treatment for this disease.

Research on alzheimer's disease has focused on two proteins: tau and Amyloid Precursor Protein (APP) (Stuart M.) and Mark (Mark P.M), Nature Medicine (Nature Medicine), 12(4), 392-. The brain of the affected individual accumulates abnormal forms of both proteins. Tau is hyperphosphorylated, while APP is cleaved by secretases to produce amyloid-beta (Α β) protein, which accumulates in the brain as plaques. Generally, in brain regions where plaque accumulates, the number of synapses decreases and neurites are destroyed. This suggests that amyloid β protein destroys synapses and neurites (Mark P.M, Nature, 430, 631-639, 2004).

In order to treat alzheimer's disease, research on pathogenesis has been actively conducted. Specifically, inhibitors of β -secretase and/or γ -secretase that produce amyloid β protein, proteases that degrade accumulated amyloid β protein, and acetylcholinesterase inhibitors that degrade acetylcholine have been intensively studied. In addition, since Alzheimer's disease is a chronic inflammatory disease associated with aging, studies have been made on the treatment of Alzheimer's disease with an inflammation inhibitor.

The amount of amyloid beta protein in the brain is determined by the balance between amyloid beta protein production and elimination reactions. Thus, if the amount of amyloid β protein removed is decreased, the amount of amyloid β protein will increase. The absence of neprilysin (NEP; an enzyme with activity to degrade amyloid beta) accelerates extracellular accumulation of amyloid (Kanaja-Ando et al, J.Biol.chem., 283(27), 19066-19076, 2008).

Abnormal neurites protruding from neuronal cell bodies are associated with neurological diseases. Examples of neurological disorders are alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania. In particular, epilepsy is caused by neuronal death and gliosis in the human hippocampus. Neuronal death results in neurite lysis. Multiple sclerosis is a chronic autoimmune disease occurring in the brain caused by abnormalities in Nogo a (neurite outgrowth inhibitory protein). Depression is a brain disorder caused by abnormalities in M6a (a protein associated with neurite outgrowth). It has been reported that in mice, by activating the signal transduction pathway that stimulates neurite outgrowth, the symptoms of mania will be alleviated.

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that differentiate into mesoblast series cells, such as osteocytes, chondrocytes, adipocytes, and myocytes; or ectodermal layers are cells, such as neurons. Recently, it has been reported that MSCs are likely to be differentiated into glia in the brain, and thus, there has been an attempt to differentiate MSCs into neurons (korean patent laid-open publication No. 10-2004-0016785, 2.25.2004).

Among MSCs, bone marrow-derived MSCs can be obtained from a patient. If the MSC is autografted, immune rejection does not occur and thus can be clinically used in patients. However, bone marrow donation is time consuming, psychologically and physiologically painful, and expensive work due to the various complex medical treatment phases required for collection of bone marrow-derived MSCs. However, since cord blood-derived MSCs can be easily obtained from the umbilical cord, the cord blood preservation industry is actively developing, and donors are easily found due to the infrastructure of cord blood, making it easy to obtain MSCs. In addition, MSCs obtained from allogeneic cord blood (allogeneic cordiblood) do not show an immune response after transplantation, thereby exhibiting immune stability.

All cited references are incorporated herein by reference in their entirety.

Disclosure of Invention

Technical problem

In order to treat a neural disease using stem cells, according to a conventional method, it is first necessary to differentiate stem cells into neurons, or it is necessary to administer stem cells together with a material that differentiates stem cells into neurons.

One or more embodiments of the present invention include a cell therapy method for a neurological disease that does not require differentiation of stem cells into neurons.

One or more embodiments of the present invention include a composition for preventing and treating a neurological disease comprising MSCs.

One or more embodiments of the invention include a method of preventing neuro-cytotoxicity caused by amyloid beta protein, preventing tau protein phosphorylation in neurons, preventing neurite damage, and inducing enkephalinase expression in neurons or microglia.

One or more embodiments of the invention include a kit for preventing neurocytotoxicity caused by amyloid beta protein, preventing phosphorylation of tau protein in neurons, preventing neurite damage, and inducing enkephalinase expression in neurons or microglia.

Technical solution

The inventors of the present invention found that when neurons or microglia treated with or without amyloid β protein are co-cultured with MSCs, MSC culture broth, or proteins contained in MSC culture broth, it is possible to prevent neurocytotoxicity, tau protein phosphorylation and neurite damage in neurons, caused by amyloid β protein, and induce expression of enkephalinase in neurons or microglia.

Advantageous effects

When neurons or microglia are co-cultured with MSCs, MCS culture solutions, proteins contained in MSC culture solutions, and/or signal transduction system stimulating factors inducing expression of the proteins, it is possible to prevent neurocytotoxicity caused by amyloid β protein, prevent phosphorylation of tau protein in neurons, induce expression of enkephalinase in neurons or microglia, and prevent neurite damage.

According to the present invention, a composition comprising MSCs, a culture solution of MSCs, a protein contained in a culture solution of MSCs, or a stimulating factor of a signal transduction system inducing expression of the protein can be used as a cell therapeutic composition effective for the prevention and treatment of a neurological disease.

In addition, a method and kit for preventing neurocytotoxicity caused by amyloid beta protein, preventing tau protein phosphorylation in neurons, preventing neurite damage, and inducing expression of enkephalinase in neurons using MSCs, MSC culture solutions, proteins contained in MSC culture solutions, and/or signal transduction system stimulating factors inducing expression of the proteins are provided.

Drawings

The above and other features and advantages of the present invention will be better understood by describing in detail exemplary embodiments thereof with reference to the attached drawings.

Figure 1 illustrates optical microscope images of live neurons untreated and treated with amyloid beta protein for 24 hours.

FIG. 2 shows a co-culture system for co-culturing amyloid beta treated neurons with human umbilical blood (UCB) -derived MSCs.

Figure 3 illustrates the results of fluorescent staining, which explains the effect of co-culture of neurons with human UCB-derived MSCs on neuronal death caused by amyloid beta protein (a β 42).

Figure 4 is a graph illustrating the percentage of dead neurons explaining the effect of co-culture of neurons with human UCB-derived MSCs on neuronal death by Α β 42.

Figure 5 illustrates the results of fluorescent staining, which explains the effect of co-culture of neurons with human bone marrow-derived MSCs on a β 42-induced neuronal death.

FIG. 6 illustrates neurons fluorescently stained using anti-phosphorylated tau antibody.

Figure 7 illustrates neurons treated with a β 42, co-cultured with MSCs and stained using immunofluorescence staining.

Figure 8 illustrates the expression of enkephalinase in neurons treated with a β 42 and co-cultured with bone marrow derived MSCs or UCB derived MSCs.

Figure 9 illustrates the expression of enkephalinase in neurons and microglia when Α β 42-treated neurons and microglia were co-cultured with MSCs.

Figure 10 is a graph illustrating the percentage of dead neurons treated with a β 42 and co-cultured with MSC-secreted proteins.

FIG. 11 is a graph illustrating neurite length in neurons cultured with A β 42 and proteins secreted by MSCs.

FIG. 12 shows the results of RT-PCR obtained using total RNA isolated from UCB-MSCs as a template after co-culturing microglia with UCB-MSCs.

FIG. 13 shows the results of protein immunoblotting (western blotting), which indicates that the expression of NEP is increased when neurons and microglia are cultured in the presence of IL-4.

Figure 14 shows images of plaques of Α β protein in brain tissue (including hippocampus and cerebral cortex) stained with Thio-S staining.

Fig. 15 is a diagram illustrating the total area of the a β patches in the image of fig. 14.

Fig. 16 shows the results of immunoblotting (immunoblotting), which indicates changes in a β protein produced in the brains of experimental mice.

FIG. 17 shows the degree of expression of NEP in brain tissues (including hippocampus and cerebral cortex) of normal mice and mice transformed to suffer from Alzheimer's disease.

FIG. 18 is a graph illustrating band intensities of NEP in FIG. 17 measured using Quantity One software (Bio-RAD).

FIG. 19 shows the degree of expression of NEP in brain tissue (including hippocampus and cerebral cortex) of mice administered with MSC and IL-4.

FIG. 20 shows NEP expression in mouse microglia cells administered UCB-derived MSCs and IL-4.

Detailed Description

According to an embodiment of the present invention, when neurons are co-cultured with Mesenchymal Stem Cells (MSCs), it is possible to prevent or repair neuronal damage caused by amyloid beta protein, while MSCs are not differentiated into neurons during co-culture, while direct contact between neurons and MSCs does not occur. Furthermore, the inventors of the present invention found that neuronal damage caused by amyloid β protein can also be prevented or repaired when co-cultured with MSC culture solution or a specific protein contained in the culture solution.

When neurons treated with amyloid beta protein 42(a β 42) at 10 micromolar (μ M) for 24 hours (Ct + a β shown in fig. 1 and 3) were compared with untreated neurons (Ct shown in fig. 3), most of the neurons treated with a β 42 died. However, if damaged nerve cells are co-cultured with cord blood (UCB) -derived MSCs, neuronal death is prevented and cell maturation is increased (Ct + Α β + MSCs in fig. 3 and 4). The effect of UCB-derived MSCs to prevent amyloid β protein-induced neuronal death was also observed in bone marrow-derived MSCs (cortex/a β/BM-MSCs in fig. 5). When cerebral cortical neurons and MSCs were co-cultured in the presence of Α β 42 for 24 hours in the same medium, the same results were obtained as shown by Ct + Α β + MSCs in figure 3. This suggests that, if neurons were co-cultured with MSCs, neurons damaged by a β 42 could be repaired and neuronal damage caused by a β 42 could be prevented.

In addition, co-culturing tau with human UCB-derived MSCs prevents phosphorylation of tau that would otherwise be rapidly phosphorylated by Α β 42 (fig. 6).

Observation of neurons using antibodies against Tubulin β III (Tubulin β III) and MAP2 (i.e., neuronal markers) revealed that in neurons treated with a β 42, the neurites were damaged and lysed due to toxicity, and the shape of the neurons was reduced. However, when neurons were co-cultured with UCB-derived MSCs, neurites remained in neurons, and neuronal differentiation and maturation was accelerated (fig. 7).

By observing the expression of enkephalinase (NEP), a protein that degrades and removes a β 42, it was found that the expression of NEP was reduced in neurons treated with a β 42. However, when neurons were co-cultured with UCB-derived MSCs, NEP expression increased at the protein level and mRNA level (a of fig. 8). B of fig. 8 illustrates neurons stained with anti-NEP antibodies. If neurons were treated with A β 42, the red-stained fraction was significantly reduced, thereby indicating a reduction in NEP expression in neurons. However, if neurons were co-cultured with MSCs, expression of NEP increased. These results were also observed in experiments performed using bone marrow-derived MSCs and UCB-derived MSCs (fig. 8C). Thus, when neural cells with or without a β 42 treatment were co-cultured with MSCs, the expression level of NEP in neural cells was increased at the mRNA and protein levels. MSCs include UCB-MSCs and BM-MSCs.

Furthermore, it was also identified that UCB-derived MSCs induce expression of NEP not only in neurons (neurons) but also in microglia called brain macrophages, and remove toxic substances accumulated in the brain, such as a β of alzheimer's disease (fig. 9).

Since the above-mentioned effects are obtained by co-culturing MSCs and neurons without direct contact between MSCs and neurons, it is considered that substances secreted from MSCs cause these effects. Proteins that are not or minimally expressed when MSCs are cultured alone, but increased expression in MSCs when neurons are co-cultured with MSCs, are analyzed. As a result, a total of 14 proteins were identified in connection with prevention of toxicity caused by a β 42 and neuronal differentiation and maturation. The 14 proteins are activin (activin A), platelet factor 4(platelet factor 4, PF4), decorin (decorin), galectin 3(galectin 3), growth differentiation factor 15(growth differentiation factor 15, GDF15), phosphatidylol proteoglycan 3 (glycoprotein 3), membrane-type frizzled-related protein (MFRP), intercellular adhesion molecule 5 (interstitial adhesion molecule 5, ICAM5), insulin-like growth factor binding protein 7(insulin-like growth factor binding protein 7, IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secretory protein (cysteine secretory protein-AA-1, SPA-induced protein 1-539), platelet-induced secretory protein (RCL 1-1, SPA-induced protein 1-2, WISP1) and progranulin. When neurons were treated with a β 42 and each of the proteins in place of MSC, neuronal death was significantly reduced and neurite length was significantly increased compared to neurons treated with a β 42 alone (fig. 10 and 11). From this point of view, the above-mentioned 14 proteins will be described in more detail.

Activin a, called inhibin β a (inhiba), is a homodimeric protein. INHBA is known to be encoded by the INHBA gene in humans. The NCBI accession number for the amino acid sequence of INHBA may be: NP-002183 (SEQ ID NO: 1).

Platelet factor 4(PF4), known as chemokine (C-X-C motif) ligand 4(chemokine ligand4, CXCL4), is a small cytokine belonging to the CXC chemokine family. The gene for human PF4 is located on human chromosome 4. The NCBI accession number for the amino acid sequence of PF4 may be: NP-002610 (SEQ ID NO: 2).

The modifier is a proteoglycan having an average molecular weight of about 90 to about 140 kilodaltons (kDa). The modifier belongs to the family of leucine-rich small-molecule proteoglycans (SLRPs) and comprises a core protein (protein core) having leucine repeats and glycosaminoglycans (GAGs) consisting of Chondroitin Sulfate (CS) or Dermatan Sulfate (DS). The NCBI accession number for the amino acid sequence of the modifier may be: NP-001911 (SEQ ID NO: 3).

Galectin 3, called soluble galactoside-binding lectin 3 (LGAL 3), is a lectin that binds to beta-galactosides. For example, the NCBI accession number for the amino acid sequence of galectin 3 may be: NP-919308 (SEQ ID NO: 4).

Growth differentiation factor 15(GDF15), known as macrophage inhibitory factor 1 (MIC 1), is a protein belonging to the transforming growth factor β superfamily (transformingrowth factor beta superfamily) that controls inflammatory pathways in wounds and cell death pathways in disease processes. For example, the NCBI accession number for the amino acid sequence of GDF15 may be: NP-004855 (SEQ ID NO: 5).

Phosphoacyl-proteoglycan 3, designated GPC3, is a protein belonging to the family of phosphatidylethanoproteoglycans. For example, the NCBI accession number for the amino acid sequence of phosphatidyl-alcoglycan 3 can be: NP-004475 (SEQ ID NO: 6). Phosphoryl alcoglycan belongs to the heparan sulfate proteoglycan (heparan sulfate) family and is attached to the cell surface by forming a covalent bond with Glycosylated Phosphatidylinositol (GPI).

For example, the NCBI accession number for the amino acid sequence of membrane-associated frizzled related protein (MFRP) may be: NP-113621 (SEQ ID NO: 7).

Intercellular adhesion molecule 5(ICAM5), known as telecephalin, belongs to the ICAM family. ICAM is a type I transmembrane glycoprotein (type I) containing 2 to 9 immunoglobulin pseudotype C2 domains and binds to leukocyte adhesion lymphocyte function-associated antigen 1 (LFA-1) protein. For example, the NCBI accession number for the amino acid sequence of ICAM5 can be: NP-003250 (SEQ ID NO: 8).

Insulin-like growth factor binding protein 7(IGFBP7) belongs to the IGFBP family that specifically binds to insulin-like growth factor (IGF). IGFBP7 is also known as IGF-binding protein-related protein 1 (IGFBP-rp 1). For example, the NCBI accession number for the amino acid sequence of IGFBP7 may be: NP-001544 (SEQ ID NO: 9).

Platelet-derived growth factor AA (PDGF-AA) belongs to the PDGF family. PDGF-AA is a homodimeric glycoprotein comprising a PDGF alpha polypeptide, referred to as two PDGFAs. PDGF is a protein that controls cell growth and differentiation. PDGF is also associated with angiogenesis. For example, the NCBI accession number for the amino acid sequence of PDGFA may be: XP-001126441 (SEQ ID NO: 10).

For example, the NCBI accession number for the amino acid sequence of cysteine-rich acidic secreted protein-like 1(SPARCL1) can be: NP-004675 (SEQ ID NO: 11).

Thrombospondin 1(TSP1) is a homotrimeric protein bound via disulfide bonds. Thrombospondin 1 is mediated by cells and matrix interactions of adhesion glycoprotein. Thrombospondin 1 can bind to fibrinogen (fibrinogen), fibronectin (fibronectin), laminin (laminin), and collagen type V (type V collagen). For example, the NCBI accession number for the amino acid sequence of thrombospondin 1 can be: NP-003237 (SEQ ID NO: 12).

WNT1 induces signaling pathway protein 1 (witp 1 inducing pathway protein 1, WISP1), referred to as CCN4, and belongs to the WISP protein subfamily and Connective Tissue Growth Factor (CTGF) family. WNT1 is a cysteine-rich glycosylated signaling protein that mediates a variety of developmental processes. CTGF family members are characterized by having four conserved cysteine-rich domains: an IGF binding domain, a type C vWF module (vWF type C module), a thrombospondin domain, and a C-terminal cystine-knot-like domain (C-terminal cystine knob-like domain). For example, the NCBI accession number for the amino acid sequence of WISP1 may be: NP-003873 (SEQ ID NO: 13).

The Progranulin (PGN) is a precursor of granulin (granulin). The progranulin is a single precursor protein with 7.5 repeats of the highly conserved 12 cysteine progranulin/epithelin (epithelin) motif, and progranulin (GRN) is cleaved from progranulin and belongs to the family of secreted glycosylated peptides. The progranulin is also known as the epithelin precursor (proepithelin) and the PC cell-derived growth factor. For example, the NCBI accession number for the amino acid sequence of the progranulin may be: NP-001012497 (SEQ ID NO: 14).

Increased expression of enkephalinase (NEP) in microglia and neurons is identified if the microglia and neurons are cultured in the presence of Interleukin-4 (Interleukin-4, IL-4). In addition, reduction of amyloid plaques is also identified if UCB-derived MSCs (UCB-MSCs) or IL-4 are administered to mice with Alzheimer's disease. Increased NEP expression in brain tissue (including hippocampus and/or cerebral cortex) was also identified if UCB-MSC or IL-4 was administered to mice with Alzheimer's disease. Increased expression of NEP in microglia in brain tissue is also identified if UCB-MSC or IL-4 is administered to mice with Alzheimer's disease.

Interleukin-4 (IL-4) is a cytokine that induces natural T helper cells (Th0 cells) to differentiate into Th2 cells. IL-4 activates Th2 cells to further produce IL-4. The NCBI accession number for the amino acid sequence of IL-4 may be: NP-000580 (SEQ ID NO: 30) or NP-067258.

These 14 proteins may include not only human-derived proteins but also mammalian-derived proteins. For example, the mammal comprises a rodent, and the rodent can comprise, for example, a mouse or a rat.

Even though recent studies on tissue regeneration medicine have suggested the possibility of using stem cells to treat neurodegenerative disorders such as alzheimer's disease, the currently available stem cell technologies have not been developed sufficiently to be applicable to extensive memory loss in the brain, such as alzheimer's disease. However, the present inventors found that MSC can reduce the cytotoxicity caused by amyloid β protein and accelerate differentiation and proliferation of neural stem cells in brain. This raises the possibility of developing cell preparations for the treatment of Alzheimer's disease and other neurological diseases. In addition, several proteins secreted by MSCs have been found to have therapeutic effects on neurological diseases such as alzheimer's disease and thus have increased the potential for preventing and treating neurological diseases.

The present invention provides a pharmaceutical composition for preventing or treating a neurological disease, comprising Mesenchymal Stem Cells (MSCs), a culture solution of MSCs, a protein contained in a culture solution of MSCs, and/or a signal transduction system-stimulating factor inducing expression of the protein. The neurological disease may be a disease caused by neurite damage. The neurological disorder can be alzheimer's disease, parkinson's disease, depression, epilepsy, multiple sclerosis, mania, or any combination thereof.

Pre-dementia syndromes (pre-dementia syndromes) exhibiting mild cognitive impairment can be diagnosed using neuropsychological tests. Approximately 12% of patients with mild cognitive impairment have been reported to progress to alzheimer's disease annually. Surprisingly, about 80% of patients with mild cognitive impairment progressed to alzheimer's disease after 6 years without any treatment. Therefore, when the pharmaceutical composition of the present invention is administered to a patient with mild cognitive impairment, it is possible to prevent or delay the progression to alzheimer's disease.

The present invention also provides methods and kits for preventing neurocytotoxicity in neurons caused by amyloid beta protein treatment, preventing phosphorylation of tau protein in neurons, preventing neurite damage, and inducing expression of enkephalinase in neurons using MSCs, MSC culture solutions, proteins contained in MSC culture solutions, or signal transduction system stimulating factors inducing expression of the proteins in vitro or in vivo. The kit may further comprise components required for culturing neurons.

The pharmaceutical composition of the present invention comprising MSC, MSC culture solution, protein contained in MSC culture solution, or signal transduction system stimulating factor inducing the expression of the protein, can be administered together with other effective ingredients having an effect of preventing or treating Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis, mania, and the like.

The pharmaceutical composition may further comprise pharmaceutically acceptable additives in addition to the active ingredient, and may be formulated into unit dose formulations suitable for administration to a patient using any of the methods known in the pharmaceutical arts. For this purpose, formulations for parenteral administration may be used, such as injection formulations or topical administration formulations. For example, formulations for parenteral administration may be used, such as sterile solution or suspension formulations for injection, where necessary with water or other pharmaceutically acceptable solvents. For example, unit dose formulations may be prepared using pharmaceutically acceptable carriers or vehicles, such as sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives, and binders.

The pharmaceutical formulation may be administered parenterally using any method known in the art. Parenteral administration can include both topical and systemic administration. Topical administration can be carried out by administering the drug formulation directly into the injured area or a region surrounding the injured area, such as the brain or spinal cord, its surrounding region, or an area opposite thereto. Systemic administration can be carried out by administering the drug formulation into the spinal fluid, vein or artery. Spinal fluid comprises cerebrospinal fluid. An artery may be a region that supplies blood to a damaged area. In addition, the administration can be carried out according to the methods disclosed in Douglas-Contzyoka (Douglas Kondziolka), Pittsburgh (Pittsburgh), Neurology (Neurology), Vol.55, p.565-. Specifically, a small hole having a diameter of 1 cm was cut in the skull of an individual, and a suspension of MSCs in Hank's Balanced Salt Solution (HBSS) was injected into the small hole using a long-needle syringe (long-needle system) and a stereotactic frame (stereotactic frame) for injecting the suspension into the correct position.

The dose range of MSC may be 1X 10 per day⁴To 1X 10⁷One cell/kg (body weight), e.g. 5X 10 per day⁵To 5X 10⁶Individual cells per kilogram (body weight), which can be administered in a single dose or in divided doses. It will be appreciated, however, that the amount of MSCs (e.g., UCB-derived MSCs) actually administered to a patient will be determined by a variety of relevant factors, including the type of disease, the severity of the disease, the chosen route of administration, and the weight, age, and sex of the individual patient.

The present invention also provides a method of preventing or treating a neurological disease in a subject, the method comprising administering to the subject a pharmaceutical composition comprising at least one selected from the group consisting of Mesenchymal Stem Cells (MSCs) and MSC culture fluid.

The administration used in the method may be topical administration or systemic administration. The pharmaceutical composition administered may be in an amount effective to prevent or treat the disease. One skilled in the art will readily appreciate that the effective amount may vary depending on the disease condition.

The pharmaceutical composition used in the method is the same as the above-described pharmaceutical composition. In the method, MSCs contained in the pharmaceutical composition may be collected not only from autologous cells, but also from allogeneic cells from other and medically experimental animals. Cells stored in frozen form may also be used. This method of treatment is not limited to humans. In general, MSCs are also applicable to mammals as well as humans.

In the method, the neurological disease may be a disease caused by at least one selected from the group consisting of amyloid beta protein, tau protein hyperphosphorylation, enkephalinase under expression, and neurite damage. The neurological disorder can be alzheimer's disease, parkinson's disease, depression, epilepsy, multiple sclerosis, or mania.

Amyloid beta protein (a β) as used herein is the major component of amyloid plaques found in the brain of alzheimer's disease patients. Amyloid beta protein (a β) may be a peptide comprising an amino acid derived from the C-terminus of Amyloid Precursor Protein (APP) as a transmembrane glycoprotein. A β can be produced from APP by the sequential operation of β -secretase and γ -secretase. For example, a β may comprise 39 to 43 amino acids, such as 40 to 42 amino acids. A.beta.may comprise residues 672-713 (A.beta.42) or 672-711 (A.beta.40) of the amino acid sequence of NCBI accession No. NP-000475 (SEQ ID NO: 19), which is the human amyloid beta A4 protein isoform precursor (APP). Amyloid beta protein (a β) may be derived from a mammal. For example, a β may be derived from a human or mouse.

As used herein, a "tau protein" is a microtubule-associated protein found in neurons of the central nervous system. tau protein interacts with tubulin, stabilizing microtubules and promoting tubulin assembly of microtubules. Brain tissue is known to contain 6 different tau isoforms. Tau protein hyperphosphorylation is known to be associated with alzheimer's disease outbreaks. tau protein is a microtubule-associated protein with high solubility. For humans, tau protein is found primarily in neurons, not non-neuronal cells. One function of tau protein is to control the stabilization of axonal microtubules. For example, the tau protein may be microtubule-associated protein tau isoform 2, the amino acid sequence of which has NCBI accession number NP-005901 (SEQ ID NO: 20). tau proteins may be derived from mammals. For example, tau protein may be derived from a human or mouse.

Enkephalinase is a zinc-dependent metalloprotease that breaks down large amounts of small secreted peptides. If amyloid beta protein undergoes abnormal misfolding and accumulates in neural tissue, enkephalinase breaks down the amyloid beta protein that causes Alzheimer's disease. For example, the NCBI accession number for the amino acid sequence of enkephalinase can be: NP-000893 (SEQ ID NO: 21). Enkephalinase may be derived from a mammal. For example, enkephalinase may be derived from a human or mouse.

The present invention also provides a method of reducing amyloid plaques in a neural tissue, the method comprising culturing the neural tissue in the presence of at least one selected from the group consisting of Mesenchymal Stem Cells (MSCs) and a culture solution of MSCs.

In the method, neural tissue, such as neurons, may be cultured in vitro or in vivo. In vitro culture can be performed in media for MSCs and/or neural tissue (e.g., neurons) as is known in the art. MSCs may or may not be in direct contact with neural tissue (e.g., neurons) when cultured. For example, culturing can be performed by separating MSCs from neural tissue (e.g., neurons) with a porous membrane. The pore size and configuration of the membrane may be large enough to allow passage of bioactive materials in the MSC medium through the pores, but not the cells. The bioactive material can be a protein, a sugar, and a nucleic acid. The membrane may be disposed such that the MSCs are cultured on the membrane and the neural tissue (e.g., neurons) are cultured under the membrane, whereby the bioactive material passes through the membrane under the force of gravity to reach under the membrane.

The in vivo culturing may further comprise administering at least one selected from the group consisting of MSCs and a culture of MSCs to the subject. Administration may be topical or systemic. An amount effective to reduce the amount of plaque may be administered. One skilled in the art will readily appreciate that the effective amount may vary depending on the disease condition. The subject may be any animal in need of reduction of amyloid plaques in neural tissue. The animal may comprise a mammal. The mammal may comprise a human, mouse or rat.

Reducing amyloid plaques in the neural tissue may be a reduction in the amount of amyloid plaques in the neural tissue compared to the amount of amyloid plaques when the neural tissue (e.g., neurons) is cultured in the absence of MSCs and MSC culture fluid.

The term "amyloid plaques" as used in the present specification may be insoluble fibrin aggregates, comprising amyloid beta protein. Amyloid plaques may be present within cells, on cell membranes and/or in the interstitial spaces between cells.

The term "neural tissue" as used herein encompasses the central nervous system, e.g. brain tissue. The brain tissue includes brain tissue and hippocampus. The brain tissue comprises the cerebral cortex. The nerve tissue includes nerve cells as well as the nerve tissue itself. The neural cells comprise neuronal cells and/or microglia. Culturing neural tissue comprises culturing neural cells, such as neuronal cells and/or microglia, in vivo or in vitro.

The present invention also provides a method of reducing the extent of phosphorylation of tau protein in a neuron, the method comprising culturing the neuron in the presence of at least one selected from the group consisting of a Mesenchymal Stem Cell (MSC) and a MSC culture solution.

The culturing is as described above with reference to the method of reducing amyloid plaques.

Reducing phosphorylation of tau protein in a neuron may be a reduction in the amount of tau protein phosphorylation compared to the amount of tau protein phosphorylation when the neuron is cultured in the absence of MSC and MSC culture.

The present invention also provides a method of increasing the expression of enkephalinase in a neuron or a microglia cell, the method comprising culturing the neuron or the microglia cell in the presence of at least one selected from the group consisting of Mesenchymal Stem Cells (MSCs) and MSC culture broth.

The culturing is as described above with reference to the method of reducing amyloid plaques in neural tissue. Increasing enkephalinase expression in a neuron or microglia may be an increase in enkephalinase expression in a neuron or a microglia as compared to the expression of enkephalinase in a neuron or a microglia when the neuron or the microglia is cultured in the absence of MSC and MSC culture medium.

The present invention also provides a method of increasing neurite outgrowth of neurons, the method comprising culturing neurons in the presence of at least one selected from the group consisting of Mesenchymal Stem Cells (MSCs) and MSC culture solutions.

The culturing is as described above with reference to the method of reducing amyloid plaques in neural tissue. The neuron may be a normal neuron or a neuron with a neurite damaged (e.g., by a β). The increased neuronal neurite outgrowth may be increased neuronal neurite outgrowth compared to neuronal neurite outgrowth when the neurons are cultured in the absence of MSC and MSC culture fluid.

The present invention also provides a method of preventing or treating a neurological disease in a subject, the method comprising administering a pharmaceutical composition comprising at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

The administration used in the method may be topical administration or systemic administration. An amount effective to prevent or treat the neurological disorder can be administered. One skilled in the art will readily appreciate that the effective amount may vary depending on the disease condition.

The pharmaceutical composition used in the method is the same as the above-described pharmaceutical composition.

In the method, the neurological disease may be a disease caused by at least one selected from the group consisting of amyloid beta protein, tau protein hyperphosphorylation, enkephalinase under expression, and neurite damage. The neurological disorder can be alzheimer's disease, parkinson's disease, depression, epilepsy, multiple sclerosis, or mania.

The present invention also provides a method of reducing amyloid plaques in a neural tissue, the method comprising culturing the neural tissue in the presence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

In the method, neural tissue, such as neurons, may be cultured in vitro or in vivo. The in vivo culturing may further comprise administering to the subject at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof. Administration may be topical or systemic. An amount effective to reduce the amount of plaque may be administered. One skilled in the art will readily appreciate that the effective amount may vary depending on the disease condition. For example, the amount administered may be from about 1 ng/kg body weight to about 100 mg/kg body weight, such as from about 10 ng/kg body weight to about 50 mg/kg body weight for each selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof. The formulation administered may further comprise additives such as water, culture medium, buffers or excipients. The subject may be any animal in need of reduction of amyloid plaques in neural tissue. The animal may comprise a mammal. The mammal may comprise a human, mouse or rat.

Amyloid plaques are reduced in the presence thereof when compared to the absence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

The invention also provides a method of reducing the extent of phosphorylation of tau protein in a neuron, the method comprising culturing the neuron in the presence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

The culturing is as described above with reference to the method of reducing amyloid plaques in neural tissue. A reduced degree of phosphorylation of tau protein in a neuron in the presence thereof when compared to the absence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

The present invention also provides a method of increasing the expression of enkephalinase in a neuron or a microglia cell, the method comprising culturing the neuron or the microglia cell in the presence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

The culturing is as described above with reference to the method of reducing amyloid plaques in neural tissue. Increased expression of enkephalinase in neurons or microglia in the presence thereof when compared to the absence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

The present invention also provides a method of increasing neurite outgrowth of a neuron, said method comprising culturing a neuron in the presence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

The culturing is as described above with reference to the method of reducing amyloid plaques in neural tissue. The neuron may be a normal neuron or a neuron with a neurite damaged (e.g., by a β). Neuronal neurite outgrowth is increased in its presence when compared to the absence of at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP7, PDGF-AA, SPARCL1, thrombospondin-1, WISP1, a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

As used herein, "Mesenchymal Stem Cells (MSCs)" may be MSCs isolated from at least one selected from the group consisting of: mammals, such as humans, embryonal yolk sac, placenta, umbilical cord, cord blood, skin, peripheral blood, bone marrow, adipose tissue, muscle, liver, neural tissue, periosteum, fetal membrane, synovium, synovial fluid, amniotic membrane, meniscus, anterior cruciate ligament (antigen ligament), articular chondrocytes, deciduous teeth, pericytes, dendritic bone (trabecular bone), infrapatellar fat pad (infrapatellar fat pad), spleen, thymus, and other tissues containing MSCs; or MSCs expanded by culturing isolated MSCs.

As used herein, "cord blood" refers to blood taken from the umbilical vein that connects the placenta of a mammal (including a human) with the body of its newborn. As used herein, "cord blood-derived MSCs" refers to MSCs isolated from cord blood of a mammal (e.g., a human) or MSCs expanded by culturing the isolated UCB-MSCs.

As used herein, "treatment" refers to: preventing the manifestation of a disease or condition that has not yet been diagnosed in an animal (e.g., a mammal, including a human being) susceptible to the disease or condition; inhibiting disease progression; or to alleviate the disease.

Terms not defined herein have the meanings commonly used in the art.

Any known method, for example, the method disclosed in korean patent No. 489248, can be used to isolate monocytes containing MSCs from cord blood. For example, a Ficoll-Hypaque diversity gradient method (Ficoll-Hypaque diversity method) can be used, but is not limited to this method. Specifically, after birth and before removal of the placenta, mononuclear cells were obtained from cord blood collected from the umbilical vein using a ficoll-diatrizoate gradient centrifugation. Monocytes were washed several times to remove impurities. The isolated monocytes may undergo MSC isolation and culture, or be frozen at very low temperatures for long term storage until use.

Any known method can be used to isolate MSC from cord blood and culture MSC (Korean patent laid-open publication No. 2003-0069115; and Pictingger MF), Science (Science), 284: 143-7, 1999; and Lazeluss HM (Lazarus HM) et al, Bone marrow transplantation (Bone MarrowTransplant), 16: 557-64, 1995).

First, the collected cord blood was centrifuged using a polysucrose-diatrizoate gradient to separate mononuclear cells containing hematopoietic stem cells (hematopoietic stem cells) and MSCs, and the mononuclear cells were washed several times to remove impurities. Monocytes were cultured in a petri dish at the appropriate density. Subsequently, the monocytes are propagated to form a monolayer. MSCs among monocytes proliferate into uniform spindle-shaped long cell colonies when observed using a phase contrast microscope (phase contrast microscope). The grown cells were repeatedly subcultured to obtain the desired number of cells.

The cells contained in the compositions of the invention may be stored in frozen form using known methods. (Campos et al, Cryobiology (Cryobiology) 35: 921-924, 1995). The medium used in the frozen form may comprise 10% dimethyl sulfoxide (DMSO) and one of 10% to 20% Fetal Bovine Serum (FBS), human peripheral blood or cord blood plasma or serum. The cells may be suspended so that about 1X 10 cells are present in 1 ml of the medium⁶To 5X 10⁶And (4) cells.

The cell suspension is dispensed into glass or plastic ampoules for deep freezing (deep freezing), and the ampoules can then be sealed and placed into a deep freezer maintained at a programmed temperature. In this regard, for example, the freezing procedure used controls the freezing rate at-1 deg.C/min, thereby minimizing damage to the cells during thawing. When the ampoule temperature is below-90 deg.C, it can be transferred to a liquid nitrogen tank and maintained at a temperature below-150 deg.C.

To thaw the cells, the ampoule is quickly transferred from the liquid nitrogen tank to a 37 ℃ water bath. The thawed cells in the ampoule are quickly placed into a culture vessel containing culture medium under sterile conditions.

In the present invention, the medium used to isolate and culture MSCs may be any medium known in the art for performing ordinary cell culture, containing 10% to 30% FBS, human peripheral blood or cord blood plasma or serum. For example, the medium can be Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), alpha-MEM, McCoys 5A medium, Eagle's basal medium, Comnart's Medical research laboratory (Connaught Medical research)Research laboratory, CMRL) medium, Glasgow minimal essential medium (Glasgow minimal essential medium), Han's F-12medium (Ham's F-12medium), Issche's Modified Dulbecco's Medium (IMDM), Libovitz's L-15medium (Liebovitz' L-15medium), or Ross Park Memorial Institute (RPMI) 1640 medium, such as DMEM. The concentration of suspended cells may be 5X 10 per 1 ml of medium³To 2X 10⁴And (4) cells.

In addition, the cell culture medium of the present invention may further comprise one or more accessory components. The auxiliary component can be fetal calf serum, horse serum or human serum; and antibiotics such as penicillin g (penicillin g), streptomycin sulfate (streptomycin sulfate), and gentamicin (gentamicin); antifungal agents, such as amphotericin b (amphotericin b) and nystatin; and mixtures thereof, to prevent microbial contamination.

Cord blood-derived cells do not express the histocompatibility antigen HLA-DR (class II), which is the major cause of rejection after tissue or organ Transplantation (Lebran K C (Le Blanc, K C), Experimental hematology (Exp Hematol), 31: 890-. Since these cells can minimize immune responses after transplantation (e.g., rejection of transplanted tissues or organs), autologous as well as allogeneic cord blood can be used. Frozen cells may also be used.

The culture solution of MSCs may be a culture solution for culturing mammalian cells such as human bone marrow-derived MSCs, UCB-derived MSCs, adipose tissue-derived stem cells, embryo yolk sac-derived MSCs, placenta-derived MSCs, skin-derived MSCs, peripheral blood-derived MSCs, muscle-derived MSCs, liver-derived MSCs, nerve tissue-derived MSCs, periosteum-derived MSCs, umbilical cord-derived MSCs, fetal membrane-derived MSCs, synovial membrane-derived MSCs, amnion-derived MSCs, meniscus-derived MSCs, anterior cruciate ligament-derived MSCs, articular chondrocyte-derived MSCs, mammary tooth-derived MSCs, pericyte-derived MSCs, dendritic bone-derived MSCs, infrapatellar fat pad-derived MSCs, spleen-derived MSCs, thymus-derived MSCs, and MSCs isolated from other tissues containing MSCs, and/or cultured MSCs.

The culture medium can be, for example, a cell culture medium containing FBS or plasma or serum of human peripheral blood or cord blood. The cell culture medium may include, for example, DMEM, MEM, alpha-MEM, McCoys 5A medium, Ito's basal medium, CMRL medium, Glasgow minimum essential medium, Han's F-12medium, Isaacs Modified Dubecard's Medium (IMDM), Leboviz L-15medium, and RPM11640 medium, but is not limited thereto.

The MSC culture solution of the present invention may comprise at least one selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP, PDGF-AA, SPARCL1, thrombospondin 1, WISP1 and a granulin precursor, IL-4 or a factor inducing at least one of said proteins.

The pharmaceutical composition of the present invention may comprise at least one protein selected from the group consisting of: activin A, PF4, a modifier, galectin 3, GDF15, phosphatidyl-alcoglycan 3, MFRP, ICAM5, IGFBP, PDGF-AA, SPARCL1, thrombospondin 1, WISP1 and a granulin precursor, IL-4 or a factor inducing at least one of said proteins.

The factor inducing at least one of the proteins may be a signal transduction system stimulating factor and any known factor. The factor may be the following example, but is not limited thereto. The galectin 3 inducing factor may include at least one selected from the group consisting of phorbol 12-myristate 13-acetate (PMA) and modified lipoprotein. It is known that PMA or lipoprotein induces galectin 3 by Protein Kinase C (PKC), mitogen-activated protein kinase 1, 2 (MAPK-1, 2) and p38 kinase. The factor inducing PDGF-AA may comprise at least one selected from the group consisting of avian erythroblastosis virus E26(avian erythroblastosis virus E26, v Ets) oncogene homolog 1(Ets-1) and lysophosphatidylcholine (lysophosphatidylcholine). Hemolytic phosphatidylcholine is known to induce PDGF-AA via MAPK-1, 2.

All cited references are incorporated herein by reference in their entirety.

The present invention will be described in more detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples of the invention

Example 1: isolation and culture of neural stem cells

Neural stem cells used herein were isolated as follows. Neural stem cells were isolated from the cerebral cortex and hippocampus of 14-day spera-Dawley rat (Sprague-Dawley rat; Orient bio inc, Korea) embryos (embryonic day 14, E14). First, the abdomen of the pregnant rat was incised, and the embryo was separated using scissors and forceps. Embryos were washed with Hank's Balanced Salt Solution (HBSS) for stripping and placed into petri dishes containing ice-cold HBSS. The cerebral cortex and hippocampus of the E14 embryo were separated under a microscope using a needle and forceps. The separated cerebral cortex was aspirated 10 to 20 times using a pipette to make it into a single cell and put into serum-free culture solution. Individual cells were treated with poly-L-ornithine (15 μ g/ml; Sigma, st. louis, MO), at 37 ℃ for 16 hours and smeared onto fibronectin (1 μ g/ml; Sigma) coated coverslips for at least 2 hours. Serum-free Neurobasal growth factor (bFGF) in serum-free supplemented with 20 ng/ml basic FGF and B-27 serum-free additive^TMThe individual cells were cultured in medium (Gibco) for about 2 to 4 days until about 70% of the bottom surface of the dish was covered with individual cells (70% to 80% confluency). bFGF was removed and neuronal cells were induced to differentiate for 4 to 6 days. In the differentiation stageIn the presence of 5% CO₂While changing the medium and B27 additive every other day, and bFGF was added thereto every day. Differentiated neurons were used in the following examples.

Example 2: isolation and amplification of UCB-derived MSCs

Immediately after birth, a sample of Umbilical Cord Blood (UCB) is collected from the umbilical vein, with maternal consent. Specifically, the umbilical vein was punctured with a 16-gauge needle attached to a UCB collection bag containing 44 ml of citrate dextrose citrate anticoagulant-1 (CPDA-1) anticoagulant (Korean Green Cross Corp., Korea) to collect UCB into the collection bag under the force of gravity. After collection, the UCB thus obtained was treated within 48 hours, and the monocyte survival rate exceeded 90%. The collected UCB was centrifuged using a polysucrose-diatrizoate gradient (density: 1.077 g/ml; Sigma) to obtain monocytes, and the monocytes were washed several times to remove impurities. The cells were suspended in minimal essential medium (α -MEM; Gibco BRL) supplemented with 10% to 20% FBS (Henkenlen (Hyclone)). Cells were introduced into minimal essential medium supplemented with 10% to 20% FBS to optimal concentration and in 5% CO₂The medium was incubated at 37 ℃ in an incubator while changing the medium twice a week. When the cultured cells form a monolayer and MSCs expanded in a spindle shape are identified using a phase contrast microscope, the cells are repeatedly subcultured so as to sufficiently expand the MSCs. UCB-derived MSCs were cultured in α -MEM supplemented with 10% to 20% FBS.

Example 3: toxicity of amyloid beta protein

To prepare for ideal conditions for Alzheimer's disease onset, serum-free Neurobasal in a serum-free manner without bFGF and B27 and containing amyloid beta protein fragments 1-42(A beta 42, Sigma, A9810) known to cause Alzheimer's disease at a concentration of 10 micromolar^TMNeurons differentiated as described in example 1 were cultured in medium. After the neural stem cells are differentiated for 3 to 4 days, they are observed with a microscopeMorphological characteristics of neural stem cells. If differentiation into neurons was identified, cells were treated with A β for 24 hours.

Figure 1 illustrates optical microscope images of live neurons untreated and treated with amyloid beta protein for 24 hours, which were used to measure morphological changes of neurons. As the concentration of amyloid β protein increases, the number of neuronal deaths increases. In FIG. 1, the control group shows serum-free Neurobasal in the absence of amyloid beta protein^TMNeurons cultured in medium, A β -1 micromolar, A β -5 micromolar and A β -10 micromolar, show neurons cultured for 24 hours in medium comprising amyloid β protein at 1 micromolar, 5 micromolar and 10 micromolar, respectively.

Example 4: effect of human UCB-derived MSCs on neuronal death by Co-culture with neurons treated with amyloid beta protein

When amyloid beta treated neurons were co-cultured with human UCB-derived MSCs, neurons damaged by toxic substances (e.g. amyloid beta protein) were observed.

Specifically, E14 embryonic cerebral cortex stem cells and hippocampal stem cells were isolated, and the isolated stem cells were proliferated and differentiated into neurons in the same manner as described in example 1, followed by treatment with amyloid β protein at a concentration of 10 μm as in example 3. After 12 hours of amyloid β protein treatment, the amyloid β protein-treated neurons were co-cultured with human UCB-derived MSCs in the presence of amyloid β protein for 12 hours, whereby the cells were cultured in the presence of amyloid β protein for 24 hours in total. Co-cultivation was carried out in the co-cultivation system shown in FIG. 2. Figure 2 shows a co-culture system for co-culturing amyloid beta treated neurons with human UCB-derived MSCs. Referring to fig. 2, co-cultivation system 100 comprises upper chamber 10 and lower chamber 40, wherein the bottom of upper chamber 10 comprises microporous membrane 30 having a pore size of about 1 micron. Human UCB-derived MSCs 20 are cultured in an upper chamber 10, and neurons 50 differentiated from cerebral cortex stem cells or hippocampal stem cells are cultured in a lower chamber 40. The upper chamber 10 and the lower chamber 40 may be separated from each other, and the lower surface of the bottom of the upper chamber 10 is spaced apart from the upper surface of the bottom of the lower chamber 40 by about 1 mm. The co-culture is performed by culturing the cells in the lower chamber 40 and the upper chamber 10 separately, and adding the upper chamber 10 to the culture medium of the lower chamber 40.

Untreated cerebral cortex and hippocampus-derived neurons, cerebral cortex and hippocampus-derived neurons treated with amyloid beta protein, and cerebral cortex and hippocampus-derived neurons not treated with amyloid beta protein and co-cultured with MSC were also cultured and observed. After amyloid β protein treatment, injured cerebral cortex and hippocampal-derived neurons were co-cultured with human UCB-derived MSCs for 24 hours, followed by observing the degree of neuronal injury using a microscope. Serum-free Neurobasal without bFGF and B27 is used^TMThe culture medium (Gibco) was used for the culture.

To quantitatively measure neuronal death caused by amyloid β treatment, live and dead cells were measured using a fluorescent staining assay. Using LIVE/DEAD against animal cells^TMThe viability/cytotoxicity assay kit (Sigma, L3224) analyzed cytotoxicity. The kit comprises calcein AM (calcein AM) for identifying living cells and ethidium homodimer (ethidium homodimer) for identifying dead cells. Calcein AM is a non-fluorescent dye that is permeable to cells and converts to green fluorescent calcein in living cells by hydrolysis of acetoxymethylester by esterases in the cells. Ethidium homodimer cannot permeate the membrane of a living cell, but permeates the damaged cell membrane, and binds to nucleic acid of the cell to emit red fluorescence.

In the lower chamber 40 of the co-culture system 100, cerebral cortex and hippocampal-derived neurons were cultured with a medium containing a β 42, so that neurons were treated directly with a β 42. By staining live/dead cells, dead cells were stained red, while live cells were stained green. As a result, when the cells treated with 10. mu. mol of A.beta.42 for 24 hours (Ct + A.beta.of FIG. 3) were compared with the untreated cells (Ct of FIG. 3), the green fluorescence was significantly reducedAnd extensive red fluorescence was observed with a β 42 treatment, thus indicating that a β 42 treatment killed most neurons. However, if the damaged neural stem cells are co-cultured with UCB-derived MSCs in the co-culture system 100 shown in fig. 2, neuronal death is prevented and neuronal maturation is increased (Ct + Α β + MSCs in fig. 3). This indicates that when Α β 42-injured neurons were co-cultured with UCB-MSCs, damaged cells could be restored. In FIG. 3, Ct + Abeta + MSC shows serum-free Neurobasal containing Abeta 42 at a concentration of 10. mu.M^TMCerebral cortical neurons were cultured in culture medium for 12 hours and then co-cultured with UCB-derived MSCs in the presence of a 10 micromolar concentration of Α β 42 for 12 hours. In addition, in the presence of 10 micromolar A.beta.42 in the lower chamber 40 in serum-free Neurobasal^TMCerebral cortex-derived neurons were cultured in the medium, and when UCB-derived MSCs were simultaneously cultured in the same medium for 24 hours in the upper chamber 10, the results were the same as those shown in Ct + Α β + MSCs of fig. 3. Therefore, if neurons are co-cultured with UCB-MSC, Α β 42-injured neurons can be recovered and Α β 42-induced injury can be prevented.

In FIG. 3, Ct is shown in Neurobasal without A β 42 serum^TM24 hours of cortical neurons cultured in culture media, Ct + Abeta was shown in serum-free Neurobasal containing Abeta 42 at a concentration of 10 micromolar^TM24 hours of cortical neurons cultured in culture media, Ct + Abeta + MSC were shown in serum-free Neurobasal containing Abeta 42 at a concentration of 10. mu.M^TMCerebral cortical neurons cultured for 12 hours in culture and subsequently co-cultured with UCB-derived MSCs in the presence of 10 micromolar A β 42 for 12 hours, and Ct + MSCs were shown in serum-free Neurobasal without A β 42^TMCerebral cortical neurons were cultured in medium for 12 hours and then co-cultured with UCB-derived MSCs for 12 hours.

Fig. 4 is a graph illustrating the percentage of dead neurons based on the results of fig. 3. In fig. 4, cortex shows the results of the control group in which cortical-derived neurons were cultured in the medium containing no a β 42, cortex + a β shows the results obtained by culturing cortical-derived neurons for 24 hours in the medium containing 10 micromolar a β 42, cortex + a β + MSC shows the results obtained by culturing cortical-derived neurons for 12 hours in the medium containing 10 micromolar a β 42 and then co-culturing the cortical-derived neurons with human UCB-derived MSCs for 12 hours in the presence of 10 micromolar a β 42, and cortex + MSC shows the results obtained by culturing cortical-derived neurons for 12 hours in the medium containing no a β 42 and then co-culturing the cortical-derived neurons with human UCB-derived MSCs for 12 hours.

Example 5: effect of human bone marrow-derived MSCs on neuronal death by Co-culture with neurons treated with amyloid beta protein

Experiments were performed in the same manner as in example 4 using bone marrow-derived MSCs (BM-MSCs) collected from donated bone marrow. When Α β -treated neurons were co-cultured with bone marrow-derived MSCs, neuronal death was prevented as in example 4 (Ct/Α β/BM-MSC in figure 5).

Figure 5 illustrates the results of fluorescent staining, which explains the effect of co-culture of neurons with human bone marrow-derived MSCs on Α β 42-induced neuronal death. In fig. 5, Ct shows the result of culturing a control group of cerebral cortical-derived neurons in a medium containing no a β, Ct + a β shows the result of culturing the cerebral cortical-derived neurons in a medium containing a β at a concentration of 10 micromolar for 24 hours, Ct/a β/BM-MSC shows the result of culturing the cerebral cortical-derived neurons in a medium containing a β at a concentration of 10 micromolar for 12 hours and then co-culturing the cerebral cortical-derived neurons with human bone marrow-derived MSCs in the presence of a β at a concentration of 10 micromolar for 12 hours, and Ct + BM-MSC shows the result of culturing the cerebral cortical-derived neurons in a medium containing no a β for 12 hours and then co-culturing the cerebral cortical-derived neurons with human bone marrow-derived MSCs for 12 hours.

Example 6: effect of Co-culture of human UCB-derived MSCs with neurons treated with amyloid beta protein on tau protein phosphorylation

Figure 6 illustrates neurons stained with an anti-phosphorylated tau antibody, an antibody that binds tau that is phosphorylated under the action of a β 42, where tau is referred to as a protein that induces neuronal death. The anti-phosphorylated tau antibody was conjugated with red fluorescent Cy3 to observe the binding of the anti-phosphorylated tau antibody to phosphorylated tau.

The first row in fig. 6 shows neurons stained with an anti-phosphorylated tau antibody conjugated with Cy3, and the second row in fig. 6 shows neurons stained with 4', 6-diamidino-2-phenylindole (DAPI). In the first row of fig. 6, Ct shows the results of the control group in which cortical-derived neurons were cultured in a medium containing no a β, a β 42 shows the results obtained by culturing cortical-derived neurons for 24 hours in a medium containing a β at a concentration of 10 micromolar, a β 42/MSC shows the results obtained by culturing cortical-derived neurons for 12 hours in a medium containing a β at a concentration of 10 micromolar and then co-culturing the cortical-derived neurons with human UCB-derived MSCs for 12 hours in the presence of a β 42 at a concentration of 10 micromolar, and MSC shows the results obtained by culturing cortical-derived neurons for 12 hours in a medium containing no a β and then co-culturing the cortical-derived neurons with human UCB-derived MSCs for 12 hours. As shown in the first row of fig. 6, tau protein is rapidly phosphorylated in neurons, but dephosphorylated by co-culture with human UCB-derived MSCs (see a β 42 and a β 42/MSC of fig. 6).

As shown in the second row of fig. 6, DAPI staining showed that cortical neurons not stained by anti-phosphorylated tau antibody were retained in the first row of fig. 6. DAPI staining was performed using VECTASHIELD^TM(vectored LABORATORIES (vectorer laboratrories)) and a blocking tablet containing DAPI (mounting medium) was added to the slide on which the cells were deposited just before they were observed using a microscope.

Example 7: analysis of differentiated neurons using immunofluorescence staining when amyloid beta treated neurons were co-cultured with human UCB-derived MSCs

Neurons derived from the cerebral cortex and hippocampus were stained with antibodies that specifically bind to microtubule-associated protein (MAP2) and tubulin β III, a known marker of neuronal differentiation.

Immunofluorescent staining was performed as follows. Neurons were fixed in wells of 12-well plates using 4% paraformaldehyde (paraformaldehyde) for 20 min at room temperature and washed 4 times with 0.1% BSA/PBS for 5 min each. Subsequently, a solution containing 10% Normal Goat Serum (NGS), 0.3% Triton X-100(Triton X-100) and 0.1% BSA/PBS was added thereto and reacted at room temperature for 30 to 45 minutes, thereby preventing non-specific reaction. A solution containing primary antibody, 10% NGS and 0.1% BSA/PBS was added to each well and reacted at 4 ℃ overnight. The resulting material was washed 3 times with 0.1% BSA/PBS for 5 minutes each. To this, a secondary antibody and a 0.1% BSA/PBS solution containing a reagent binding to the secondary antibody were added and reacted for 4 minutes, followed by washing the resultant 4 times with 0.1% BSA/PBS for 5 minutes each. Primary antibodies were prepared by diluting monoclonal anti-tubulin beta III antibodies (Sigma) and rabbit anti-tubulin-related protein (MAP)2 polyclonal antibodies (Chemicon) produced in mice in buffer solutions at 1: 500 and 1: 200, respectively. The secondary antibody was prepared by diluting biotinylated anti-mouse antibody and biotinylated anti-rabbit antibody (Vector) in buffer at 1: 200, respectively. The reagent for binding to the secondary antibody is prepared by diluting dichlorotriazinyl fluorescein (DTAF; Jackson ImmunoResearch) in a buffer solution at a ratio of 1: 200.

In neurons treated with a β 42 (brain and hippocampal-derived neurons), neurite lysis was caused due to toxicity, and the shape of the neurons was reduced. On the other hand, in neurons co-cultured with UCB-derived MSCs, neurites were maintained and neuronal maturation was accelerated (A, B and C of fig. 7).

Figure 7 illustrates neurons treated with a β 42, co-cultured with UCB-derived MSA, and stained using immunofluorescence staining with anti-tubulin β III and anti-MAP 2 and subjected to western immunoblotting.

Fig. 7 a shows cortical-derived neurons, and fig. 7B shows hippocampal-derived neurons. MAP2 and tubulin β III show the results obtained by immunofluorescence staining using anti-MAP 2 and anti-tubulin β III, respectively. Control group was shown in Neurobasal without serum containing A.beta.^TMResults of a control group in which cortical neurons or hippocampal neurons were cultured for 24 hours in a medium, Abeta 42 shows the results obtained by culturing cortical neurons or hippocampal neurons for 24 hours in a medium containing Abeta at a concentration of 10. mu.M, and Abeta 42/MSC shows the results obtained in serum-free Neurobasal cells containing Abeta at a concentration of 10. mu.M^TMA result obtained by culturing the cortical-or hippocampal-derived neurons in a culture medium for 12 hours and then co-culturing the cortical-or hippocampal-derived neurons with human UCB-derived MSCs in the presence of a β 42 at a concentration of 10 μmolar for 12 hours, and MSCs show a result obtained by culturing the cortical-or hippocampal-derived neurons in a culture medium containing no a β for 12 hours and then co-culturing the cortical-or hippocampal-derived neurons with human UCB-derived MSCs for 12 hours.

Fig. 7C shows the results of co-culturing cerebral cortical-derived neurons treated with Α β 42 with UCB-derived MSCs and western immunoblotting of the co-cultured neurons with anti-MAP 2 antibody. First, in a lysis buffer containing Sodium Dodecyl Sulfate (SDS), a membrane of a neuron is disrupted using an ultrasonicator to extract a protein. The extracted proteins were subjected to electrophoresis using SDS-polyacrylamide gel to separate the proteins according to size. When the electrophoresis was terminated, the protein was transferred to a nitrocellulose membrane using its electrical properties and reacted with an anti-MAP 2 antibody (Millipore chem) diluted in PBS containing 3% skim milk. Subsequently, an anti-rabbit antibody (Vector) conjugated with dichlorotriazinyl fluorescein (DTAF, Jackson immuno Research) conjugated with streptavidin (streptavidin) was added thereto, and the resultant was treated with an Enhanced Chemiluminescence (ECL) substrate solution, followed by developing the resultant using an X-ray film. In fig. 7C, the control, a β + MSC and MSC are the same as described above. In C of FIG. 7, 200 represents a molecular weight marker of 200 kilodaltons.

Example 8: induction of enkephalinase expression in neurons and microglia by human UCB-derived MSCs

Enkephalinase (NEP) and Insulin Degrading Enzyme (IDE) are well-known proteins that degrade a β 42 in vivo. Furthermore, it has been reported that NEP knockdown in mice causes Alzheimer's disease symptoms. The neurons prepared in examples 4 to 7 were collected and lysed to extract proteins. Proteins were separated using electrophoresis in SDS-PAGE and expression of the proteins was measured by western blotting of the separated proteins using anti-enkephalinase antibodies. In addition, NEP-specific primers were used to measure NEP mRNA expression by RT-PCR. In addition, the cultured cells were stained with an anti-NEP antibody.

First, neurons were fixed in wells of a 12-well plate using 4% paraformaldehyde for 20 minutes at room temperature, and washed 4 times with 0.1% BSA/PBS for 5 minutes each. Subsequently, a solution containing 10% Normal Goat Serum (NGS), 0.3% Triton X-100 and 0.1% BSA/PBS was added thereto at room temperature for 30 to 45 minutes, thereby preventing non-specific reaction. 10% NGS containing primary antibody and 0.1% BSA/PBS were added to each well and reacted overnight at 4 ℃. The resulting material was washed 3 times with 0.1% BSA/PBS for 5 minutes each. To this, a secondary antibody and a 0.1% BSA/PBS solution containing a reagent binding to the secondary antibody were added, and reacted at room temperature for 40 minutes, and the resultant was washed 4 times with 0.1% BSA/PBS for 5 minutes each. Monoclonal anti-NEP antibodies (Sigma) produced in mice and diluted 1: 500 with buffer solution were used as primary antibodies. Biotinylated anti-mouse antibody (Vector) diluted 1: 200 in buffer was used as secondary antibody. Streptavidin conjugated dichlorotriazinyl fluorescein (DTAF, Jackson Immuno Research) diluted 1: 200 in buffer was used as reagent for binding to secondary antibodies.

Figure 8 illustrates the expression of enkephalinase in rat neurons treated with Α β 42 and co-cultured with human bone marrow derived MSCs or human UCB derived MSCs.

In fig. 8 a, the upper panel shows western immunoblot analysis of cultured rat cerebral cortical derived neurons. Neurons displayed in Neurobasal serum-free, A β -free^TMThe results of the control group in which rat cortical neuron was cultured for 24 hours in the medium, neuron + A.beta.were shown in the results obtained by culturing rat cortical neuron in the medium containing A.beta.at a concentration of 10. mu.M for 24 hours, and neuron + A.beta. + MSC were shown in the serum-free Neurobasal containing A.beta.at a concentration of 10. mu.M^TMRat cortical-derived neurons were cultured in a medium for 12 hours and then co-cultured with human UCB-derived MSCs in the presence of a 10 micromolar concentration of Α β for 12 hours, and neuron + MSCs showed results obtained by culturing rat cortical-derived neurons in a medium without Α β for 12 hours and then co-culturing rat cortical-derived neurons with human UCB-derived MSCs for 12 hours.

In A of FIG. 8, the lower panel shows the RT-PCR results obtained using mRNA isolated from cultured rat neurons as a template. PCR primers specific for the rat NEP gene (SEQ ID NOS: 15 and 16) and specific for the β -actin gene (SEQ ID NOS: 17 and 18) were used. According to RT-PCR, an amplified NEP gene (422 base pairs) and an amplified β -actin gene (300 base pairs) were generated. Neurons, neuron + Α β + MSC and neuron + MSC are as described above.

As shown in a of fig. 8, if rat neurons were treated with a β 42, expression of NEP decreased. If rat neurons treated with Α β 42 were co-cultured with human UCB-derived MSCs, the expression of NEP increased at the protein and mRNA level. This suggests that human MSC stimulates rat neurons to increase NEP production and remove toxic a β 42 protein.

In B of fig. 8, Ct, a β + MSC and MSC correspond to neuron, neuron + a β + MSC and neuron + MSC, respectively.

Cells were stained according to the following method. First, neurons were fixed in wells of a 12-well plate using 4% paraformaldehyde for 20 minutes at room temperature, and washed 4 times with 0.1% BSA/PBS for 5 minutes each. Subsequently, a solution containing 10% Normal Goat Serum (NGS), 0.3% Triton X-100 and 0.1% BSA/PBS was added thereto at room temperature for 30 to 45 minutes, thereby preventing non-specific reaction. 10% NGS containing primary antibody and 0.1% BSA/PBS were added to each well and reacted overnight at 4 ℃. The resulting material was washed 3 times with 0.1% BSA/PBS for 5 minutes each. To this, a secondary antibody and a 0.1% BSA/PBS solution containing a reagent binding to the secondary antibody were added, and reacted at room temperature for 40 minutes, and the resultant was washed 4 times with 0.1% BSA/PBS for 5 minutes each. Monoclonal anti-NEP antibodies (Sigma) produced in mice and diluted 1: 500 with buffer solution were used as primary antibodies. Biotinylated anti-mouse antibody (Vector) diluted 1: 200 in buffer was used as secondary antibody. Streptavidin conjugated dichlorotriazinyl fluorescein (DTAF, Jackson Immunoresearch) diluted 1: 200 in buffer was used as reagent for binding to secondary antibodies.

As shown in B of fig. 8, if neurons were treated with a β 42, the red-stained portion was significantly reduced, thereby indicating a reduction in expression of NEP in the neurons. However, if neurons were co-cultured with MSCs, expression of NEP was restored.

FIG. 8C shows the results of RT-PCR, which indicates that the use of bone marrow-derived MSCs (BM-MSCs) increases NEP expression in rat neurons.

RT-PCR of NEP and β -actin was performed under the same conditions using the same primers as described for a of fig. 8. In FIG. 8C, lane 1 shows serum-free Neurobasal without A β^TMResults of control group in which rat cerebral cortical neurons were cultured for 24 hours in culture medium, lane 2 andlane 3 shows the results obtained by culturing rat cortical neurons in a culture medium without Α β for 12 hours and then co-culturing the rat cortical neurons with human bone marrow-derived MSCs (BM-MSC1 and BM-MSC2) for 12 hours. In this regard, BM-MSC1 and BM-MSC2 represent cells obtained from different donors. The results shown in C of fig. 8 demonstrate that when rat cortical-derived neurons were co-cultured with human BM-MSCs, NEP expression of rat cortical-derived neurons was increased at the mRNA level. In addition, according to western blot analysis and immunoblot analysis, it was confirmed that when rat cerebral cortical derived neurons were co-cultured with human BM-MSCs, the expression of NEP in neurons was increased at the protein level.

The brain contains not only neurons but also microglia, which are called macrophages of the brain and can remove toxic substances accumulated in the brain. Microglia remove a β from alzheimer's disease. According to recent reports, a decrease in NEP expression in microglia will accelerate the progression of alzheimer's disease. Therefore, immunofluorescence staining was used to identify the repair effect of human UCB cells on NEP expression in neurons and microglia (fig. 9). Figure 9 illustrates the expression of enkephalinase in neurons and microglia when neurons treated with a β 42 were co-cultured with MSCs.

FIG. 9 first row shows serum-free Neurobasal containing A.beta.at a concentration of 10 micromolar^TMCultured in a medium for 12 hours, followed by co-culture with human UCB-derived MSCs for 12 hours in the presence of a 10 micromolar concentration of Α β 42, and double-stained cortical-derived neurons using antibodies that specifically bind to each of the neuronal markers MAP2 and NEP. Staining was performed in the same manner as in B of fig. 8, except that for MAP2, a rabbit anti-MAP 2 antibody was used as a primary antibody, a biotinylated anti-rabbit antibody was used as a secondary antibody bound to the primary antibody, and streptavidin-conjugated dichlorotriazinyl fluorescein (DTAF, Jackson immuno Research) was used as a reagent bound to the secondary antibody; while a monoclonal anti-NEP antibody (Sigma) produced in mice against NEP was used as a primary antibody, biotinylated anti-NEPA mouse antibody (Vector) was used as a secondary antibody, and dichlorotriazinyl fluorescein conjugated with streptavidin (DTAF, Jackson immuno Research) was used as a reagent that binds to the secondary antibody. In the first row of fig. 9, MAP2 and NEP show neurons stained with anti-MAP 2 antibody and anti-NEP antibody, respectively, and MAP2+ NEP shows overlapping images of neurons stained with anti-MAP 2 antibody and anti-NEP antibody, respectively. DAPI shows the results obtained using DAPI staining in the same manner as in the second row of fig. 6.

Since both MAP2 and NEP showed stained cells as shown in the first row of fig. 9, both MAP2 and NEP were identified as being expressed in neurons. Further, according to the image overlap (MAP2+ NEP), MAP2 and NEP were found in the same region, and thus it was identified that both MAP2 and NEP were expressed. DAPI was used to stain neurons and thereby identify neurons as remaining normal.

The second row of fig. 9 shows the results of the same experiment as shown in the first row of fig. 9, but using microglia instead of neurons and using the microglia markers CD40 and NEP as markers for microglia instead of MAP2 and NEP. For CD40, CD40 was stained with goat anti-CD 40 antibody as a primary antibody, biotin conjugated anti-goat antibody as a secondary antibody bound to the primary antibody, and streptavidin conjugated dichlorotriazinyl fluorescein (DTAF, Jackson immuno Research) diluted 1: 200 in buffer solution as a reagent bound to the secondary antibody.

Since both CD40 and NEP showed stained cells as shown in the second row of fig. 9, both CD40 and NEP were identified as being expressed in microglia. Furthermore, from the image overlap (CD40+ NEP), CD40 and NEP were found in the same region, and thus it was identified that MAP2 and NEP were both expressed in microglia. The microglia were stained with DAPI and thus identified as being normal.

According to the results of the first and second rows of fig. 9, if neurons and microglia were co-cultured with UCB-derived MSCs, the expression of NEP in a β -treated neurons and microglia was induced.

Example 9: identification of proteins secreted by MSCs that prevent Α β 42 toxicity and validation of the role of said proteins

From the results of examples 4 to 8, it was identified that if neurons treated with a β 42 were co-cultured with MSCs without direct contact between the neurons and MSCs, a β 42 toxicity in the neurons was inhibited. It is predicted that the interaction between the substance secreted by MSC and neuron inhibits the toxicity of a β 42.

In example 9, substances secreted by MSCs that inhibit a β 42 toxicity were detected and identified.

(1) Detection of MSC-derived substances inhibiting Abeta 42 toxicity

First, cells were cultured under various conditions.

Culture group 1: in serum-free Neurobasal without A beta^TMCerebral cortex derived neurons were cultured in the medium for 24 hours.

Culture group 2: serum-free Neurobasal containing A beta at a concentration of 10 micromolar^TMCerebral cortex derived neurons were cultured in the medium for 24 hours.

Culture group 3: serum-free Neurobasal containing A beta at a concentration of 10 micromolar^TMCerebral cortical neurons were cultured in medium for 12 hours, followed by co-culture with human UCB-derived MSCs in the presence of 10 micromolar Abeta for 12 hours.

Culture group 4: serum-free Neurobasal containing A beta at a concentration of 10 micromolar^TMHuman UCB-derived MSCs were cultured in medium for 24 hours.

Culture groups 5 and 6: in serum-free Neurobasal^TMHuman UCB-derived MSCs were cultured in medium for 24 hours.

Subsequently, the culture media of the culture groups 1 to 6 were collected, and cytokines and proteins were analyzed and compared with each other to detectCytokines or proteins that are not or are rarely expressed when stem cells are cultured alone but are increased when stem cells and neurons are co-cultured. Using RayBio^TMCytokine analysis was performed on a Human Cytokine Antibody Array I G series (Human Cytokine Antibody Array I G series; RayBiotech, Inc), using RayBio^TMProtein analysis was performed on human cytokine Antibody Array I G series/Biotin-labeled Antibody Array I G series (Biotin Label Based Antibody Array I Geries; Lebei Taike Co.). 54,504 proteins can be analyzed using these two arrays.

Comparing the data from these analyses, proteins were selected that were not or minimally expressed when stem cells were cultured alone, but increased when stem cells were co-cultured with neurons. The following 14 proteins were thus identified:

activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1 (TSP1), wnt-1-induced secreted protein 1(WISP1), and granule protein Precursor (PGN).

It is estimated that these 14 proteins inhibit the toxicity of neurons treated with a β and promote neuronal differentiation and maturation.

(2) Identification of the Activity of the 14 proteins detected

The 14 proteins tested were purchased from (R)&D systems Co Ltd (R)&D SYSTEMS)). Subsequently, cortical neurons were treated with A β and contained 25 ng/ml activin A, 25 ng/ml PF4, 3 ng/ml galectin 3, 100 ng/ml modifier, 50 ng/ml GDF15, 50 ng/ml phosphatidylol proteoglycan 3, 50 ng/ml MFRP, 50 ng/ml ICAM5, 30 ng/ml IGFBP7, 50 ng/ml PDGF-AA, and 50 ng/ml CPP, respectivelySerum-free Neurobasal of g/ml SPARCL1, 50 ng/ml TSP1, 50 ng/ml WISP1 and 50 ng/ml particle protein precursor^TMCultured in the medium for 24 hours. Then, by using LIVE/DEAD^TMViability/cytotoxicity assay kit (Sigma, L3224) was fluorescence stained to measure neuronal death. The degree of cell death caused by a β was calculated based on the number of dead cells and live cells. Cell death was calculated using the ratio of the number of dead cells to the total number of cells.

Figure 10 is a graph illustrating the percentage of dead neurons treated with a β 42 and co-cultured with MSC-secreted proteins. In FIG. 10, the cortex is shown in serum-free Neurobasal without A β 42^TM24-hour cerebral cortical neurons cultured in culture medium, cortical + Abeta displayed in serum-free Neurobasal containing Abeta 42 at a concentration of 10 micromolar^TM24-hour cerebral cortical neurons cultured in culture medium, cortical + Abeta + MSC were shown in serum-free Neurobasal containing Abeta 42 at a concentration of 10 micromolar^TMCerebral cortical neurons cultured in culture medium for 12 hours and subsequently co-cultured with UCB-derived MSCs in the presence of 10 micromolar A.beta.42 for 12 hours, and cortical + MSCs were shown in serum-free Neurobasal without A.beta.42^TMCerebral cortical neurons were cultured in medium for 12 hours and then co-cultured with UCB-derived MSCs for 12 hours. A.beta.is shown in serum-free Neurobasal containing A.beta.42 and 14 proteins at each of the above concentrations^TMCortical neurons cultured in culture or 24 hours (p < 0.03 and p < 0.01 in FIG. 10, respectively, indicate error ranges for t-test of less than 3% and 1%, respectively).

As shown in fig. 10, each of the 14 proteins inhibited neuronal death caused by a β 42. The degree of inhibition of cell death is in descending order: cortex + Α β + MSC, galectin 3, WISP1 and MFRP. This indicates that co-culture with MSCs, i.e. the combination of 14 proteins, had the strongest effect on inhibition of Α β toxicity.

To measure the effect of the protein on neuronal maturation, the length of neurites in cultured cells was measured. Neurons were cultured under the same conditions as described with respect to fig. 10. 100 cells were randomly selected from each culture group, and neurite length was measured using i-solution software (iM technologies).

FIG. 11 is a graph illustrating neurite length in neurons cultured with A β 42 and proteins secreted by MSCs. In FIG. 11, the culture group was the same as that described in FIG. 10, and the neurite length was the average length. As shown in fig. 11, each of the 14 proteins or the combination of 14 proteins resulted in a significant increase in neurite length compared to neurons treated with a β 42.

Example 10: identification of cytokines secreted by MSC that induce enkephalinase expression in microglia

The co-cultivation system 100 described in example 4 is used herein. Microglia cells (BV2) were cultured in the lower chamber 40 and UCB-derived MSCs (UCB-MSCs) were cultured in the upper chamber 10. BV2 cells were immortalized cells prepared by infecting mouse microglia with a v-raf/v-myc recombinant retrovirus and exhibited the trait of activating microglia. In the lower chamber 40, BV2 cells were cultured in DMEM supplemented with 5% FBS, UCB-derived MSCs cultured in α -MEM supplemented with 5% FBS were added to the upper chamber 10, and the medium was replaced with serum-free DMEM, thereby performing co-culture. Cells were co-cultured in serum-free DMEM for 24 hours. Then, MSCs were collected from the upper chamber 10, and total RNA was obtained using triazole (trizol) reagent, followed by RT-PCR using the total RNA as a template. Primers for amplifying IL-4(SEQ ID NOS: 22 and 23), IL-6(SEQ ID NOS: 24 and 25), IL-8(SEQ ID NOS: 26 and 27) and monocyte chemotactic protein-1 (MCP-1; SEQ ID NOS: 28 and 29) genes were used. Primers (SEQ ID NOS: 17 and 18) were used to amplify beta-actin as a control group. In the control group, UCB-derived MSCs (UCB-MSCs) cultured under the same conditions as described above were used, but the UCB-derived MSCs were not co-cultured with microglia (BV 2).

FIG. 12 shows the results of RT-PCR obtained using total RNA isolated from UCB-MSCs as a template after co-culturing microglia with UCB-MSCs. As shown in FIG. 12, if microglia cells were co-cultured with UCB-MSCs, the expression of IL-4, IL-6, IL-8 and MCP-1 in UCB-MSCs was increased.

Microglia, BV2 cells, neurons, and SH-SY5Y cells (ATCC) were cultured in the presence of IL-4, IL-6, IL-8, and MCP-1, respectively, followed by collection of BV2 cells and SH-SY5Y cells. The collected cells were lysed, and the proteins were separated from the lysate according to size, and the resultant was subjected to western blotting using an anti-NEP antibody. As a result, NEP expression increased over time in BV2 cells and SHY-5Y cells cultured in the presence of IL-4 when compared to in the absence of IL-4. SH-SY5Y cells are neuroblastoma derived from three clones of SK-N-SH. SH-SY5Y cells represent neuronal cells.

FIG. 13 shows the results of Western immunoblotting, which indicates that the expression of NEP is increased when neurons and microglia are cultured in the presence of IL-4. FIG. 13A shows the results of Western immunoblotting of microglia (BV2 cells) cultured in DMEM containing 10 ng/ml IL-4 for 24 hours. B of FIG. 13 shows the results of Western immunoblotting of neurons (SH-SY5Y cells) cultured in alpha-MEM containing 10 ng/ml IL-4 for 24 hours.

Example 11: reduction of amyloid plaques (thioflavin S staining and immunoblotting) by administration of UCB-derived MSCs into hippocampus and cortex of mice transformed to suffer from alzheimer' S disease

To improve the therapeutic effect, PBS containing 1X 10 was applied using a stereotactic frame⁴PBS of individual UCB-derived MSCs and PBS containing 200. mu.g/kg (body weight) IL-4 (Peprotech) were administered to the hippocampus of 10-month-old mice transformed to suffer from Alzheimer's disease. After 10 days, mice were sacrificed and brain tissue was collected from their hippocampus and cerebral cortex. The resulting brain tissue was sliced and stained with thiosulfate (Sigma) to identify amyloid β plaques. To identify plaques, brain tissue was reacted for 5 min with a solution of thioflavin (sigma) dissolved in 50% ethanolA clock. After the reaction, the thin slices of brain tissue were washed with 50% ethanol and water for 5 minutes. These slices were observed using a fluorescence microscope to identify amyloid plaques in brain tissue.

Figure 14 shows images of amyloid β plaques in brain tissue (including hippocampus and cerebral cortex) stained with Thio-S staining. As shown in fig. 14, amyloid β plaques were significantly reduced in the culture group administered with UCB-derived MSCs and IL-4. In FIG. 14, PBS, MSC and IL-4 show the culture groups to which PBS, UCB-derived MSC and IL-4 were administered, respectively.

Fig. 15 is a graph illustrating the total area of amyloid β plaques in the image of fig. 14. The area was measured using metamorphho software (Molecular devices). As shown in fig. 15, amyloid β plaques were significantly reduced in the culture group administered with MSC and IL-4 when compared with the control group.

Fig. 16 shows the results of immunoblotting, which indicates changes in amyloid β protein produced in the brain of experimental mice. The graph of fig. 16 is obtained according to the following method. First, proteins were extracted from brain tissues of mice (including hippocampus and cerebral cortex), and treated with an ultrasonicator (Branson) under the above conditions. Subsequently, the extracts were separated according to size using electrophoresis. The separated proteins were transferred to a nitrocellulose membrane by means of a potential difference, and immunoblotting was performed using an antibody capable of specifically detecting a β 42. The protein was stained using Coomassie blue (Coomassie blue; lower panel). As shown in fig. 16, the amount of a β 42 protein was significantly reduced in the culture group administered with MSC and IL-4 when compared with the culture group administered with PBS. In FIG. 16, Litter represents litters of transformed mice, and APP/PS1 mice represent mice transformed to have Alzheimer's disease. In addition, PBS, MSC and IL-4 show the culture groups to which PBS, MSC and IL-4 were administered, respectively.

Example 12: effect of UCB-derived MSCs and IL-4 on NEP expression

(1) Expression of NEP in brain tissue of Normal animals and animals transformed to suffer from Alzheimer's disease

Brain tissues of normal mice and mice transformed to have alzheimer's disease, which were bred for 6, 9, 12 and 18 months, respectively, were obtained, and proteins were extracted in the same manner as in example 11 and separated using electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and reacted with an anti-NEP antibody (R & D systems) to analyze expression of NEP.

FIG. 17 shows the degree of expression of NEP in brain tissues (including hippocampus and cerebral cortex) of normal mice and mice transformed to suffer from Alzheimer's disease. As shown in fig. 17, expression of NEP was reduced in brain tissue of mice transformed to suffer from alzheimer's disease. In FIG. 17, Litter and APP/PS1 mice are the same as described for FIG. 16. Further, lanes 6, 9, 12 and 18 show culture groups (M: month) cultured for 6, 9, 12 and 18 months, respectively.

FIG. 18 is a graph illustrating band intensities of NEP in FIG. 17 measured using Quantity One software (Bio-RAD). The band intensities are relative intensities. As shown in fig. 18, the expression of NEP was reduced in the brain tissue of the mouse transformed to have alzheimer's disease, compared to the normal mouse.

(2) Effect of UCB-derived MSCs and IL-4 on NEP expression

Mixing PBS with 1 × 10⁴PBS of UCB-derived MSCs and PBS containing 200. mu.g/kg (body weight) IL-4 (Peprotech) were administered to the hippocampus of 10-month-old mice transformed to suffer from Alzheimer's disease. After 10 days, mice were sacrificed and brain tissue including hippocampus and cerebral cortex was collected. Proteins were extracted from each brain tissue and separated using electrophoresis to analyze the amount of NEP expressed using immunoblotting.

FIG. 19 shows the degree of expression of NEP in brain tissue (including hippocampus and cerebral cortex) of mice administered with MSC and IL-4. Staining was performed using coomassie blue (lower panel). As shown in fig. 19, when compared with the normal mice shown in the above procedure (1), the expression of NEP was decreased in the cultured group administered with PBS, and the expression of NEP was similar to that of the normal mice in the cultured group administered with UCB-derived MSC and IL-4.

Example 13: effect of UCB-derived MSCs and IL-4 on NEP expression in microglia

In example 8, it has been identified that NEP is overexpressed in neurons and microglia when the neurons and microglia, respectively, are co-cultured with MSCs.

In example 13, this effect was identified in an animal model. Hippocampal tissue of the culture group administered PBS, UCB-derived MSC and IL-4 described in example 12 was stained in the same manner as shown in B of fig. 8. anti-NEP and anti-CD 40 antibodies were used as microglial markers (Santacruz Biotechnology), and the results were combined. In the anti-NEP antibody staining, the secondary antibody and the reagent binding to the secondary antibody were the same as described in example 8. In the anti-CD 40 antibody staining, the secondary antibody and the reagent bound to the secondary antibody were also the same as described in example 8.

FIG. 20 shows NEP expression in mouse microglia cells administered UCB-derived MSCs and IL-4. As shown in fig. 20, NEP overexpression in microglia was induced when UCB-derived MSCs and IL-4 were injected into animal models.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Sequence listing

Claims (29)

1. A pharmaceutical composition for preventing or treating a neurological disease comprising at least one selected from the group consisting of a mesenchymal stem cell and a culture solution of the mesenchymal stem cell.

2. The pharmaceutical composition of claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells isolated from at least one tissue having mesenchymal stem cells selected from the group consisting of: human embryo yolk sac, placenta, umbilical cord, cord blood, skin, peripheral blood, bone marrow, adipose tissue, muscle, liver, nervous tissue, periosteum, fetal membrane, synovial fluid, amniotic membrane, meniscus, anterior cruciate ligament, articular chondrocyte, deciduous teeth, pericytes, dendritic bone, infrapatellar fat pad, spleen, and thymus; and/or mesenchymal stem cells expanded from said mesenchymal stem cells.

3. The pharmaceutical composition of claim 1, wherein the mesenchymal stem cells comprise cord blood-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells.

4. The pharmaceutical composition of claim 1, wherein the culture fluid comprises at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

5. The pharmaceutical composition of claim 1, wherein the neurological disease is a disease caused by at least one selected from the group consisting of: amyloid beta plaque formation in neural tissue, tau protein phosphorylation in neurons, neurite damage, decreased enkephalinase expression in neurons, and any combination thereof.

6. The pharmaceutical composition of claim 1, wherein the neurological disease comprises at least one selected from the group consisting of: alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania.

7. A pharmaceutical composition for preventing or treating a neurological disease, comprising at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

8. The pharmaceutical composition of claim 7, wherein the neurological disease is a disease caused by at least one selected from the group consisting of: amyloid beta plaque formation in neural tissue, tau protein phosphorylation in neurons, neurite damage, decreased enkephalinase expression in neurons, and any combination thereof.

9. The pharmaceutical composition of claim 7, wherein the neurological disease comprises at least one selected from the group consisting of: alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania.

10. A kit for preventing neurocytotoxicity caused by amyloid beta protein, comprising at least one selected from the group consisting of mesenchymal stem cells and a culture solution of the mesenchymal stem cells.

11. A kit for preventing tau protein phosphorylation in a neuron comprising at least one selected from the group consisting of a mesenchymal stem cell and a culture of said mesenchymal stem cell.

12. A kit for inducing enkephalinase expression in neurons and/or microglia comprising at least one selected from the group consisting of mesenchymal stem cells and a culture of said mesenchymal stem cells.

13. A kit for preventing neuro-cytotoxicity caused by amyloid beta protein, comprising at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

14. A kit for preventing phosphorylation of tau protein in a neuron, comprising at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

15. A kit for inducing enkephalinase expression in neurons and/or microglia comprising at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

16. A method of preventing or treating a neurological disease in a subject, the method comprising administering to the subject a pharmaceutical composition comprising at least one selected from the group consisting of mesenchymal stem cells and a culture of the mesenchymal stem cells.

17. The method of claim 16, wherein the neurological disease is a disease caused by at least one selected from the group consisting of: amyloid beta plaque formation in neural tissue, tau protein phosphorylation in neurons, neurite damage, decreased enkephalinase expression in neurons, and any combination thereof.

18. The method of claim 16, wherein the neurological disease comprises at least one selected from the group consisting of: alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania.

19. A method of preventing or treating a neurological disease in a subject, the method comprising administering to the subject a pharmaceutical composition comprising at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

20. The method of claim 19, wherein the neurological disease is a disease caused by at least one selected from the group consisting of: amyloid beta plaque formation in neural tissue, tau protein phosphorylation in neurons, neurite damage, decreased enkephalinase expression in neurons, and any combination thereof.

21. The method of claim 19, wherein the neurological disease comprises at least one selected from the group consisting of: alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania.

22. A method of reducing amyloid plaques in a neural tissue by culturing the neural tissue in the presence of at least one selected from the group consisting of mesenchymal stem cells and a culture of the mesenchymal stem cells.

23. A method of reducing the extent of phosphorylation of tau protein in a neuron by culturing the neuron in the presence of at least one selected from the group consisting of a mesenchymal stem cell and a culture of the mesenchymal stem cell.

24. A method of increasing the expression of cellular enkephalinase by culturing the cells in the presence of at least one selected from the group consisting of mesenchymal stem cells and a culture of said mesenchymal stem cells, wherein the cells are at least one selected from the group consisting of neuronal cells and microglia.

25. A method of increasing neurite outgrowth of a neuron by culturing said neuron in the presence of at least one selected from the group consisting of a mesenchymal stem cell and a culture solution of said mesenchymal stem cell.

26. A method of reducing amyloid plaques in a neural tissue by culturing the neural tissue in the presence of at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

27. A method of reducing the degree of phosphorylation of tau protein in a neuron by culturing the neuron in the presence of at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.

28. A method of increasing the expression of enkephalinase in a cell by culturing the cell in the presence of at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof, wherein the cell is at least one cell selected from the group consisting of a neuronal cell and a microglia cell.

29. A method of increasing neurite outgrowth of a neuron by culturing said neuron in the presence of at least one selected from the group consisting of: activin A, platelet factor 4(PF4), a modifier, galectin 3, growth differentiation factor 15(GDF15), phosphatidylacylol proteoglycan 3, membrane frizzled-related protein (MFRP), intercellular adhesion molecule 5(ICAM5), insulin-like growth factor binding protein 7(IGFBP7), platelet-derived growth factor-AA (PDGF-AA), cysteine-rich acidic secreted protein (SPARCL1), thrombospondin-1, wnt-1induced secreted protein 1(WISP1), a granulin precursor, IL-4, a factor inducing expression thereof, and any combination thereof.