[1]The present invention is a neural precursor cell; relates to ischemic diseases or neurological inflammatory disease therapeutic composition containing the secretion proteome of (Neural precursor cell NPC) as an active ingredient.
[2]
BACKGROUND
[3]
The multipotent stem cells because of their, considered a promising candidate for the treatment of various diseases. For example, mesenchymal stem cells (mesenchymal stem cells, MSCs) can be easily obtained and separation and secrete a number of trophic factors that promote angiogenesis and inhibit inflammation (Non-Patent Document 1). These properties of MSC has been considered in the study for application in the treatment of human disease. Recent studies of the MSC has been found to contribute to many animal models and in human clinical tissue restoration therapy (Non-patent Documents 2 and 3). Some reports are the bits that MSC in neurons (Non-Patent Document 4), and stellate cells (astrocyte) (Non-Patent Document 3), but mentions the differentiation ability of the nerve series containing, these differentiated cells in vivo no in there is no clear evidence as to whether the function. The beneficial effects of the MSC cells replacement seems to be induced by, rather than side secretion (paracrine) mechanism, whereby transplants (transplantation) of MSC is likely to have, transient and limited effect than the improvement that is long-standing (non-patent document 5) .
[4]
In contrast, embryonic stem cells (embryonic stem cells, ESCs) are capable of differentiating into all the specific cell types derived from three embryonic germ lobe (embryonic germ layer), who was known to have a powerful ability to play. Of interest is, the neural progenitor cells derived from the ESC (neural precursor cells, NPCs) are primarily neurons, astrocytes and oligodendrocytes brain tissue recovery because differentiate into a specific type of neural-based cells including (oligodendrocyte) noteworthy cells are considered to be won (cell source) for. These cells also secrete some factor that promotes survival and proliferation of endogenous neural precursor cells (Non-Patent Document 6). However, a neural progenitor cell differentiation in the ESC; (Neural precursor cell NPC) or a neural precursor cell of is still not known how the culture medium is that the contribution to the improvement of the function after transplantation in a disease model.
[5]
Through diyang a method such as inverse differentiated stem cells is around multipotential (pluripotency) transgenic for not holding body cells to / transcription factor introduction, the compounds (chemical) treatment, growth factor processing through the artificial de-differentiation process of the former multipotential also known as: (induced Pluripotent stem cell iPSC) (non-Patent Document 7) refers in words iPS cells cells were induced to. These cells are showing when compared to embryonic stem cells, cell types, cultivation methods, growth rate, gene expression and chromosome mutation pattern, I multipotent, very high similarity, etc. teratoma-forming ability of the immune-deficient mice. Differentiation ability of iPS cells is not known whether the contribution to the improvement of transplantation in the NPC or the NPC disease model of how the culture is similar and differentiated from their stem cell function.
[6]
[7]
This has a number of patents and publications are referenced and its cited throughout the specification. The disclosures of the cited patents and publications are incorporated by references into this specification in its entirety is described in the two-level more apparent and content of this invention in the art to which this invention pertains.
[8]
[9]
[Prior art document]
[10]
Non-patent literature;
[11]
1. Caplan, A. I., & Dennis, J. E. (2006). Mesenchymal stem cells as trophic mediators. Journal of Cellular Biochemistry, 98, 1076-1084.
[12]
2. Chen, J., Li, Y., Katakowski, M., et al. (2003). Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat. Journal of Neuroscience Research, 73, 778-786.
[13]
3. Kopen, G. C., Prockop, D. J., & Phinney, D. G. (1999). Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proceedings of the National Academy of Sciences, 96, 10711-10716.
[14]
4. Bae, K. S., Park, J. B., Kim, H. S., Kim, D. S., Park, D. J., & Kang, S. J. (2011). Neuron-like differentiation of bone marrowderived mesenchymal stem cells. Yonsei Medical Journal,52,401-412.
[15]
5. Cho, S. R., Kim, Y. R., Kang, H. S., et al. (2009). Functional recovery after the transplantation of neurally differentiated mesenchymal stem cells derived from bone barrow in a rat model Functional recovery after the transplantation of spinal cord injury. Cell Transplantion, 18, 1359-1368.
[16]
6. Capone, C., Frigerio, S., Fumagalli, S., et al. (2007). Neurosphere- derived cells exert a neuroprotective action by changing the ischemic microenvironment. PLoSOne, 7, e373.
[17]
7. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861-872.
[18]
Detailed Description of the Invention
SUMMARY
[19]
The inventors have studied efforts to develop a fundamental method for the treatment of ischemic disease and nerve inflammation. As a result, stem cell transplantation and is a different approach, the neural progenitor cells (neural precursor cell; NPC) ischemia by reducing the ischemic area via the administration to the affected area of ​​the secretion proteome (secretome), restored the nervous system diseases and that can be treated effectively by identifying the nerve degenerative diseases and neurological diseases caused by inflammation, thereby completing the present invention.
[20]
Accordingly, it is an object of this invention to provide a composition for ischemic diseases or neurological inflammatory diseases.
[21]
Another object of the present invention to provide a method for preventing or treating ischemic diseases or neurological inflammatory disease.
[22]
[23]
Other objects and advantages of the invention will become apparent from the following detailed description, claims and drawings of the invention described below.
[24]
Problem solving means
[25]
According to one aspect of the invention there is provided a neural precursor cell; provides an ischemic disease or neurological inflammatory disease therapeutic composition containing the secretion proteome of (neural precursor cell NPC) as an active ingredient.
[26]
[27]
The inventors have studied efforts to develop a fundamental method for the treatment of ischemic disease and nerve inflammation. As a result, stem cell transplantation and is degenerative, such as a different approach, through the administration to the affected area of ​​the neural precursor cell secretion proteome to reduce the ischemic lesion, by restoring the nerve function and nerve diseases caused by ischemic diseases and inflammation it was confirmed that to effectively treat neurological diseases.
[28]
"Secretion proteome (secretome) of the neural progenitor cells", the term, as used herein, refers to an aggregate of secreted from neural progenitor cells during culture of neural progenitor cells to the outside (culture medium) protein.
[29]
According to one embodiment of the present invention, neural progenitor cells for use in the production of secreted proteome of the invention it is differentiated from the former multipotential (all-around) stem cells.
[30]
A generic name of the term "stem cells" are undifferentiated cells of the previous step is differentiation (differentiation) in each cell constituting the tissue used in the present specification, the ability to differentiate into specific cells by a specific differentiation stimulus (environment) Have. Stem cells are cell division, unlike the differentiated suspended cells can be produced (self-renewal) for the same cell as itself by cell division, when subjected to differentiation stimuli and differentiate into specific cells such differentiation is another environment or another it is characterized with flexibility (plasticity) of the differentiation, which may also be differentiated into a variety of cells by a differentiation stimulus.
[31]
Stem cells used in the present invention the bits are indefinite proliferation in, around the multipotential (all-round) (pluripotent stem cell) stem cells that can differentiate into a variety of cells derived from the three kinds of all germ layers (ectoderm, mesoderm and endoderm) to be. More specifically, the former multipotential (all-around) stem cells are embryonic stem cells (embryonic stem cell; ESCs), induced pluripotent stem cells (iPSC; induced pluripotent stem cells), embryonic germ cells (embryonic germ cell) or embryonic tumor cells ( an embryonic carcinoma cell).
[32]
Embryonic stem cells are derived from the inner cell mass (ICM) of blastocysts, embryonic germ cells are derived from primordial germ cells of the reproductive ridge (gonadal ridge) of 5-10 weeks of age.
[33]
Induced pluripotent stem cells (iPSC) is a non-one around the multipotential cell (e.g., somatic cells) before the artificially derived by inserting a specific gene which imparts omnipotence from multipotential stem cells. IPS cells have the full multipotential stem cells (e.g., in terms of having a stem cell gene and protein expression, chromosomal methylation, the doubling time (doubling time), embryoid body formation, teratoma formation, viable chimera formation, hybridization properties and minute Mars It believed to be the same as the example, embryonic stem cells).
[34]
According to one embodiment, the neural progenitor cells (neural precursor cell; NPC) neural formed by inducing differentiation of I multipotential (all-around), stem cells (e.g., embryonic stem cells or iPS cells) into nerve series cell rosette (neural rosette) is a step forward and backward in a neural precursor cell.
[35]
According to one embodiment, the neural precursor cells are PSA-NCAM (poly-sialylated neural cell adhesion molecule) - positive neural progenitor cells, neural progenitor cells; a (Neural precursor cell NPC).
[36]
According to another embodiment, the neural precursor cells are PSA-NCAM (poly-sialylated neural cell adhesion molecule) - voice neuro progenitor neural progenitor cells; a (Neural precursor cell NPC).
[37]
The PSA-NCAM- positive or negative neural progenitor cells (Neural precursor cell; NPC) may be isolated using anti--PSA NCAM- antibody in a neural rosette differentiation through a neural differentiation stimulus from around the multipotential stem cells. "Neural rosette (neural rosette)," the term refers to the neural stem cells in the early stage of neuronal differentiation of human embryonic stem cell, a neural rosette has a radial shape of the columnar. The neural rosette consists of cells expressing the early neuroectodermal (neuroectodermal) marker such Pax6 and Sox1, can differentiate into a variety of neuronal cells and glial cells.
[38]
Said neural differentiation stimulus is the method implemented in a conventional in the art, for example, serum-free medium (Tropepe V et al, Direct neural fate specification from embryonic stem cells:. A primitive mammalian neural stem cell stage acquired through a default mechanism . Neuron 30:. 6578 (2001)), FGFs (fibroblast growth factors), treatment of the parent reportage Gen (morphogens), such as Wnt and RA (retinoic acid) (Ying QL et al Conversion of embryonic stem cells into neuroectodermal precursors in. . adherent monoculture Nat Biotechnol 21:., but can be differentiated by 183 186 (2003)), and the like.
[39]
To the antibody may be a monoclonal antibody to polyclonal or monoclonal. Antibodies to PSA-NCAM is the method implemented in a conventional in the art, for example, the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (U.S. Patent No. method No. 4,816,56) or a phage antibody library (Clackson et al, Nature, 352: 624-628 (1991) Biol, and Marks et al, J. Mol, 222:.. 58, by 1-597 (1991)) It can be prepared. The general process for manufacturing antibodies is Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, and described in detail in 1991, supra, are incorporated by reference herein. For example, the production of hybridoma cells producing monoclonal antibodies to non-apoptotic screen cell lines antibody made by combining and producing lymphocytes, skills required for this process are well known to those skilled in the art can be easily performed. A polyclonal antibody and injected into a suitable animal for PSA-NCAM antigen, and collecting anti-serum from the animal, and then,
[40]
As used herein, the term "antibody" is used, referring to the PSA-NCAM is an antigen-binding fragment of an antibody molecule as an antibody, and binding specifically to the PSA-NCAM protein, as well as the complete antibody forms of the PSA-NCAM It includes. A complete antibody is a fragment that has two full length light chains and two full length structure and antigen-binding fragment is an antigen-binding function of each light chain is linked to a heavy chain and a disulfide bond, an antibody molecule having a heavy chain of the It includes Fab, F (ab '), F (ab') 2 and Fv.
[41]
PSA-NCAM- using antibody-positive neural progenitor cells (Neural precursor cell; NPC) for the separation, fluorescence-activated cell sorter (fluorescence-activating cell sorters: FACS), magnetic activated cell sorter (magnetic activated cell sorter: MACS) and complement-mediated dissolution (complement-mediated lysis) methods can be used.
[42]
According to one embodiment of the present invention, the secretion proteome is a form containing neural progenitor cells in the cell culture medium obtained by culturing the animal cell culture medium. That is, in compositions of the invention can include a culture of neural progenitor cells that contain the secretion proteome of neural progenitor cells.
[43]
According to one embodiment, the neural progenitor cells (neural precursor cell, NPC) of the cell culture medium is serum-free animal with a neural precursor cell containing ITS (insulin / transferrin / selenium) and bFGF (basic fibroblast growth factor) after incubation in cell culture medium can be obtained by removing the cells. The culture, the subculture a neural precursor cell, e.g., N2, B-27 and / or the addition of a Gem21 bFGF- containing cells has four or more passages repeated in a culture medium obtained by culturing a neural precursor cell can be used.
[44]
Removal of the cells in the culture medium can be carried out using conventional cell separation methods such as centrifugation, filtration.
[45]
In the cell culture medium it can be used without limitation, a conventional culture medium used during culture of neural progenitor cells, for example, may be DMEM / F12.
[46]
According to one embodiment, the neural precursor cells; and (Neural precursor cell NPC) will differentiated from the human induced pluripotent stem cell (iPSC), secretion proteome of the neural precursor cell comprises a protein of the following:
[47]
Oh Green (Agrin), annexin A5 (Annexin A5), BSG (Basigin), bigeulri Khan (Biglycan), Carl ponin -3 (Calponin-3), Cocteau god-like proteins (Coactosin-like protein), Kofi Lin - 1 (Cofilin-1), collagen alpha-2, tuberculin -3 (Cullin-3), des trim (Destrin), a distributor glycans (Dystroglycan), F. Lin -B2 (Ephrin-B2), tin Expo-2 ( Exportin-2), Lin Eze (Ezrin), fibronectin, blood Dublin -1 (Fibulin-1), Frizzled-related protein, gelatin-binding protein -3 (Galectin-3 binding protein), granules Lin (Granulins), growth / differentiation factor 11 (Growh / differentiation factor 11), haptoglobin (haptoglobin), hepo peksin (Hemopexin), High mobility group protein B2, No. nerin (Hornerin), imported tin -9 (Importin-9), insulin-like growth factor binding protein 2 (Insulin-like grwoth factor-binding protein 2), lupus La protein (lupus La protein), macrophage migration inhibitory factor (macrophage migration inhibitory factor), mid-Keen (Midkine), Moe Shin (Moesin), Neuro pilrin 2 in (Neuropilin 2), play Haute (Pleiotrophin), propyl Lin -1 (Profilin-1), protein DJ-1 (Protein DJ-1), La diksin (Radixin), Secreted frizzled-related protein-2, septin -11 (Septin-11), Tallinn - 1 (Talin-1),
[48]
According to one embodiment, the neural precursor cells; and (Neural precursor cell NPC) will differentiated from human embryonic stem cells (ESC), the secretion proteome of the neural precursor cell comprises a protein of the following:
[49]
Oh green (Agrin), Annexin A2 (Annexin A2), art raktin (Attractin), bigeulri compartment (Biglycan), plasmin to serul (Ceruloplasmin), nose Lin -1 (Cofilin-1), collagen alpha-1, the nose Ronin -1X (Coronin-1X), when the dummy Dean (Dermicidin), DERP12, F. Lin -B3, cytosine exo -2 (Exostosin-2), Lin Eze (Ezrin), gel Larkin -3-binding protein, granules Lin (Granulins ), growth / differentiation factor 11 (Growh / differentiation factor 11), haptoglobin (haptoglobin), hemo peksin (Hemopexin) High mobility group protein B2, No. nerin (Hornerin), insulin-like growth factor binding protein 2 (insulin -like grwoth factor-binding protein 2), the loop number La protein, mid Keen (Midkine), Moe Shin (Moesin), multiple epidermal growth factor-like domain protein 8 (multiple epidermal growth factor-like domains protein 8), you Togendai -1 (Nidogen-1), para thymosin (Parathymosin), lean profile -2 (Profilin-2), the protein DJ-1 (protein DJ-1), secreted frizzled-related protein-2, sikeuri sat Yes Nin (Secretogranin ), Tallinn -1 (Talin-1) , Thymosin beta -4 (Thymosin beta-4), TGFBI (Transforming grwowth factor-beta-induced protein ig-h3), trans jelrin (Transgelin) and non mentin (Vimentin).
[50]
As used herein, the term "treatment" includes: (a) a disorder, inhibition of the development of the disease or condition; (B) relieving the disease, the disease or condition; Or (c) it means to remove the diseases, disorders or conditions. The compositions of the present invention serves to suppress, or remove or alleviate this the development of symptoms of ischemic disease or neurological inflammatory disease. Thus, the compositions of the present invention that can by itself be the ischemic disease or composition for the treatment of neurological inflammatory disease, or other anti-are administered in combination with inflammatory composition may be applied as a treatment adjuvant for these diseases - ischemia / anti. Thus, the term "treating" or "treatment" herein includes the meaning of "secondary treatment" or "treating adjuvants."
[51]
The term "ischemic disease" as used herein, the disease leading to a decreased blood flow due to the blood leakage (blood leakage), embolism (embolism) or infarction (infarction) of the vascular blood supply is cut off tissue necrosis He says.
[52]
According to one embodiment of the invention, the compositions of the invention curable ischemic disease is selected from ischemic heart disease, myocardial infarction, angina pectoris, not arterial ischemic diseases, ischemic diseases and ischemic limb end the group consisting of brain disease.
[53]
The terms used in the specification, the "ischemic heart disease" is damage the coronary arteries that supply blood to the heart, or are narrowed or occluded means diseases caused by decreased blood flow to the heart muscle. More specifically, in the compositions of the invention curable ischemic heart disease is selected from the group consisting of angina pectoris, myocardial infarction and heart failure.
[54]
The terms used herein, "ischemic cerebrovascular disease" means a disease or cerebral vascular damage, which is narrowing or blockage due to damaged brain tissue did not receive a blood supply. More specifically, the ischemic cerebrovascular disease is the stroke, the stroke include cerebral hemorrhage and cerebral infarction.
[55]
The term, "nervous inflammatory diseases" as used herein refers to disorders resulting from damage to the nervous system caused by inflammatory reactions, and, more specifically, refers to nerve degenerative diseases and nervous system disorders caused by inflammatory reactions.
[56]
According to one embodiment of the present invention, possible nerve inflammatory diseases treated with the compositions of the present invention is Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, diffuse Lewy body disease, white matter encephalitis It is selected from the group consisting of temporal lobe epilepsy, and spinal cord injury. Spinal cord injury in the present invention is used herein to mean including both an inflammatory spinal cord injury, and traumatic spinal cord injury.
[57]
According to still another aspect of the invention, human-derived and ESC- iPSC- derived neural progenitor cells; when administered secretion (Neural precursor cell NPC) proteomic in experimental models of stroke (pMCAo model), upon single dose and multidose PBS group and was induced by both significantly ED-1 positive cells decreased and GFAP-positive cells in comparison (see Fig. 8a-8b and 9a-9b). That is, the secretion proteome will reduce the inflammatory response and microglia (microglial cell) the fire activates (decrease) were (Fig. 8a-8b), astrocytes inactivating (astrocyte) (reduction) (Fig. 9a-9b).
[58]
In addition, the secretion proteome of the invention increase the expression of double-cor tin (doublecortin) expressed by the DCX gene and, thereby increasing the positive neural progenitor cells (see Fig. 10). Double cor tin is a neuronal protein movement (migration Neuronal protein). These results show that due to the administration of the secretion proteome is movable stem neural regeneration in the brain tissue (endogenous neural stem cell).
[59]
Sikineunde secretion proteome of the present invention increase the rate of α-SMA positive vessels, which means that the neovascular growth (see Fig. 11).
[60]
Such anti-inflammatory, up and increase new blood vessels in the stem cell intrinsic effects are neural progenitor cells of the present invention suggest that the (Neural precursor cell NPC) derived secretion proteome that the neuroprotective action (neuroprotective effect) function. That is, the neural precursor cell-derived secretion proteome of the present invention enables the treatment of the neurological inflammatory disease by showing the effect of the above.
[61]
On the other hand, before release of multipotent stem cell-derived neural precursor cell proteome of the present invention it has a therapeutic effect on spinal cord injury. According to one embodiment of the invention, the BBB test using the spinal cord injury model showed excellent recovery behavior around the multipotential stem cells (ESC) secretion proteome treated group (injection) of the derived neural progenitor cells (see Fig. 12). Induced pluripotent stem cell (iPSC) is around the multipotential stem cells from the surface with a stem cell gene and protein expression, chromosomal methylation, the doubling time (doubling time), embryoid body formation, teratoma formation, viable chimera formation, hybridization properties and minute Mars therefore it considered to be the same as (e.g., embryonic stem cells) have a therapeutic effect on spinal cord injury as well as above.
[62]
"It is administered" The term, "administration", or as used herein, refers to such that the same amount is formed in the body of a subject by direct administration to the composition of the therapeutically effective amount of the object of the present invention. Accordingly, the term "administering" is therefore includes injecting the secretion proteome of neural progenitor cells (neural precursor cell, NPC) an active ingredient of the present invention to the affected area, term "administering" is synonymous with "injection is" It is used to.
[63]
"Therapeutically effective amount" of a composition is intended to include the means to extract a sufficient amount to provide to the subject to be administered a composition for therapeutic or prophylactic effect, and this "prophylactically effective amount". The terminology used herein, "object", without limitation including human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkeys. Specifically, an object of the invention is a human.
[64]
When the compositions of the present invention is made of a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. As commonly used at the time of the carrier preparation a pharmaceutically acceptable to be contained in the pharmaceutical compositions of this invention, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, saline, PBS (phosphate buffered including a saline) or the culture medium but not limited to.
[65]
The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, preservative etc. in addition to the above components. Carriers and formulation with appropriate pharmaceutically acceptable are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
[66]
The pharmaceutical compositions of this invention may be administered orally or parenterally, specifically, parenteral administration, more specifically, intramuscularly (intramuscular) administration, the ventricle (intracerebroventricular) administration, wounds (injury) site of administration, the spinal cord or spinal (Intrathecal) or intravascular administration (intravascular) is administered. When the administration blood vessel may be administered in the artery or vein.
[67]
A suitable dose of the pharmaceutical composition of the invention may vary depending on pharmaceutical formulation methods, various prescribed by the patient's age, body weight, sex, diseased condition, food, administration time, administration route, excretion rate and response sensitivity factors such as It can be.
[68]
The pharmaceutical compositions of the invention may be administered as a single or multidose. For example, to a human or other animal may be administered as a multidose once or in divided doses. Single dose compositions may fill the daily dose to contain the amount corresponding to the amount of or a portion of the predetermined range. That is, the treatment compositions according to the present invention, or to a patient in need of treatment administered in a daily single or method of administering a plurality of times, a period of time (hours, day, week, etc.) singly or method of administering a plurality of times at an interval of to be administered.
[69]
The term, "for one-time administration" refers to the use of a composition to be administered as a composition or one-off dosage forms containing the secretion proteome of a one-time dose, and "multidose for administration" as used herein, the secretion proteome of multidose capacity It means the use of a composition to be administered as a composition or multidose form comprising.
[70]
According to still another aspect of the invention, the ischemic lesions decreased if the one-time administration of a neural progenitor cell (neural precursor cell, NPC) derived secretion proteome of the invention the stroke animal model, and showed the behavior improvement effect (FIG. 1 to see Fig. 4).
[71]
Meanwhile, the neural progenitor cells of the present invention, even if administered multidose the (Neural precursor cell NPC) derived secretion proteome stroke animal model also reduces the ischemic lesion, and exhibited a behavior improvement effect (FIG. 6a to 6i and 7 Reference). In particular, were exhibited a higher therapeutic effect in multidose improvement group the behavior compared to single administration group (see Fig. 8a-8b and 9a-9b) (Fig. 6a to 6i), it showed even higher anti-inflammatory effect, the secretion proteome the repeated administration neural endogenous stem cells and was confirmed by increasing significantly the capital of neovascularization.
[72]
The pharmaceutical compositions of the present invention is prepared in unit dosage form by formulated, using carriers and / or excipients that are pharmaceutically acceptable according to a method that can be carried out to facilitate self having ordinary skill in the art the instant invention by or in the intergranular dose container can be made. The formulations are oil or a solution in an aqueous medium, suspension, syrup or emulsion form, or may be a X, powders, powders, granules, tablets or capsule form and may comprise a dispersion agent or a stabilizer additionally.
[73]
[74]
According to another aspect of the invention there is provided (a) a neural precursor cell (neural precursor cell; NPC) a therapeutically effective amount of a proteome derived secretion; And (b) a composition comprising a carrier (carrier) a pharmaceutically acceptable; provides the prevention or treatment of ischemic diseases or neurological inflammatory disease comprises the step of administering to a subject (subject) for.
[75]
Since the method of the invention using the composition of the present invention described above, the common descriptions between them are omitted in the description in order to avoid undue redundancy leading to the complexity of this specification.
[76]
Effects of the Invention
[77]
A summary of the features and advantages of the present invention is as follows:
[78]
(I) the present invention is a neural precursor cell; provides an ischemic disease or neurological inflammatory disease therapeutic composition containing the secretion proteome of (Neural precursor cell NPC) as an active ingredient.
[79]
(Ii) secretion of neural progenitor cells of the present invention, proteomics is the anti-inflammatory and neovascularization play, intrinsic stem cells up and multiply such a role, not only reduces the ischemic damage area, bar ischemic diseases to restore nerve function and inflammation as the treatment of degenerative neurological diseases such as diseases caused by nerve damage, it can be used.
[80]
(Iii) In addition, release of the neural progenitor cells of this invention is a proteomic multidose very excellent improvement over the one-time acting effect if administered.
[81]
Brief Description of the Drawings
[82]
1 is a PBS control, medium control and human embryonic stem cells (ESC) derived neural progenitor cells (neural precursor cell;; NPC) of shows the size of the ischemic lesion site of the secretion proteome treatment group.
[83]
Figure 2 shows the PBS control, medium control and secretion weight change of proteomic treatment group.
[84]
Figure 3 shows the behavior analysis of the PBS control, medium control and secretion proteome treatment group. Figure 3a is a beam balance test results, Figure 3b is an acquisition possible traction test results, and Fig. 3c is a foot fault test result, Figure 3d shows the line cross (line cross) results showing that the activity of acting per unit of time.
[85]
Figure 4 shows the improved overall behavior of a neural effect PBS control, medium control and secretion proteome treatment group (mNSS) analysis.
[86]
In Figure 1 to 4 * P value <0.05, ** P value <0.01.
[87]
5 is a human iPS cells (iPSC) derived-neural progenitor cells; represents the experimental schedule for verifying the effect of the multidose secretion (Neural precursor cell NPC) derived proteome. BrdU was injected to verify the operation (mobilization) of the endogenous neural stem cells (enogenous neurla stem cell) present in the brain tissue using the neural precursor cell-derived secretion proteome.
[88]
Figure 6a to 6i are pluripotent stem cells derived from multipotent I-neural progenitor cells (Neural precursor cell; NPC) a single dose of a proteome derived secretion vs. 4th indicates the result of the administration. 6a, (modified Neural Sibiarity Score) mNSS; 6b, the upper body posture (torso twisting) test (left); 6c, the upper body posture inspection (torso twisting) (right); 6d, a foot fault test (foot-fault test); .; 6e, a cross line (line cross) inspection; 6f, Rearing inspection; 6g, the beam balance (balance beam) inspection; 6h, it can capture traction tests (Prehensile Traction test); 6i, illustrates the weight change of the control group and the treatment group secretion proteome. Rearing animals is a common behavior when browsing the curious from normal animals to say the attitude that stood by holding the hind paw, the unit will display the number of times per hour.
[89]
Figure 6a in to 6i White: control, black: medium control group, Yellow: secretion proteome per treatment group (secretome-S), Red: secretion proteome fourth treatment group (secretome-M), * P value <0.05, * * P value <0.01, *** P <0.001.
[90]
Figure 7 shows the size of the ischemic lesion site of the PBS control group, the medium control group, secretion proteome single dose treatment group (Secretome-S) and the secretion proteome multidose treatment group (Secretome-M).
[91]
Figure 8a-8b is a neural progenitor cell (neural precursor cell; NPC) - shows that causes a repetition of the secretion proteome derived administration (Secretome-M) were significantly ED-1 positive cells reduced the. Scale bar: 200 μm, * P <0.001.
[92]
Figure 9a-9b are neural progenitor cells (neural precursor cell; NPC) - shows that causes a repetition of the secretion proteome derived administration (Secretome-M) significantly decreased the GFAP-positive cells. Scale bar: 200 μm, * P <0.05 and *** P <0.001.
[93]
10 is a neural progenitor cell (neural precursor cell; NPC) - shows the operation of the endogenous neural stem cells in the rat brain caused by repeated administration (Secretome-M) of the derived secretion proteome (endogenous neural stem cell). Scale bar: 500 μm.
[94]
11 is a neural progenitor cell (neural precursor cell; NPC) - shows that increasing the number of repeated administration is significantly α-SMA positive vessels of origin secretion proteome. * P <0.005.
[95]
Figure 12 is an excellent recovery behavior in the secretion proteome treated group (injection) time by performing at least 8 weeks BBB test in spinal cord injury model as compared with the untreated control group shows a vision (P <0.05).
[96]
Mode for the Invention
[97]
The present invention will be described in further detail with reference to the following examples. These embodiments will be evident in the person of ordinary skill that for illustrating only the present invention in more detail, the scope of the present invention according to the aspects of the present invention is not limited by these Examples in the art .
[98]
[99]
Example
[100]
Materials and test method
[101]
Human ESC -derived or human iPSC -derived NPC and PSA- NCAM obtained positive NPC cells
[102]
The use of human cells, was approved by the Medical Research Ethics Committee (Institutional Review Board, IRB No. 4-2008-0643 ). For neural induction, each embryo derived from hESC and iPSC on hESC medium (Invitrogen) with no bFGF body (embryoid bodies, EBs) of 5 μM DM (dorsomorphin) (Sigma , St. Louis, MO) and 5-10 μM SB431542 (Calbiochem, San Diego, CA) in suspension 4 days incubation, after 20 ng / ㎖ in 1X N2 (Invitrogen) the bFGF supplemented medium Mart rijel and including the coated dish (BD Biosciences, Bedford, MA) for 5 days was adsorbed (Kim, DS, Lee, DR, Kim, HS, et al. (2012). Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and iPSC-derived neural rosettes. PLoSOne, 7, e39715). A neural rosette shown in the center of the adsorbed EB colony was separated carefully from the surrounding flat cell by using a glass pipette. Seeding a small rosette loaf Mart rijel to the coated dish, and cultured at 1X N2, DMEM / F12 with 1X B27 (Invitrogen) supplemented with (Kim, DS, Lee, JS , Leem, JW, et al. (2010). enhancement of neural differentiation from human ES Robust and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Reviews and Reports , 6, 270-281).
[103]
80 - 90% confluency the neural progenitor cells in the expanded neural rosette at the time; exposed for 1 hour to 10 μM Y27632 (Sigma) after obtaining the (Neural precursor cell NPC) that occurs long before cell death into the MACS step It was prevented. Accutase after separation using (Invitrogen), the cells (~ 1 X 10 8 cells) with 1% BSA and blocking in PBS that contains, along with micro-beads are bonded, wherein -PSA-NCAM antibody (Miltenyi Biotec) at 4 ℃ and incubated for 15 minutes. Positive, which put back the intensive washing, the cell suspension (magnetic activated cell sorting) MASC left in the column-eluted cells to a labeled tube. Separate NCAM- PSA-positive neural progenitor cells (Neural precursor cell; NPC) a N2B27 medium or 20 ng / ㎖ bFGF is added NBG medium (1XN2, 0.5XB27 0.5XG21 and replacement) (GeminiBio-Products, WestSacramento, CA) to 4-5 X 10 5 cells / cm 2 were plated again at a concentration. The culture medium was replaced every day, cells were sub-cultured every 2-3 days.
[104]
[105]
All human multipotent embryonic stem cells (ESC) derived from the separation of the secreted proteome (secretome) in neural progenitor cells (NPCs)
[106]
A human before multipotent embryonic stem cells (ESC) derived neural progenitor cells (PSA-NCAM- positive neural progenitor cells) obtained in the main culture medium (DMEM / F-12) N2 (100X- final concentration of 1X), B-27 (50X- 0.5X final concentration) and the serum removed Gem21 into supplements (50X- final concentration of 0.5X), bFGF repeated at least 4 passages in (20 ng / ㎖) was added to the Matrigel (Matrigel) coated 60 mm dish the cultured cells are then cultured to amplify and then chilled to about 90% in 8 to 10 of the dish. After removing the culture solution washed three times with PBS, and serum-free culture medium to a base (DMEM / F12) was added only ITS (100X- final concentration of 1X) and bFGF (20 ng / ㎖) were incubated for 24 hours. The control group, the culture medium of the same composition in the same amount without a cell dish (added in an amount such as the ITS and bFGF in primary culture) was added to, was used a number of times and then incubated for 24 hours in a culture medium as a control. After collection of all of the culture medium to remove cell debris, such as by centrifugation of the (30 minutes eseo 800 g) and then immediately frozen in a freezer of -70 ℃ was used after thawing, if necessary.
[107]
[108]
Human ago multipotent induced pluripotent stem cells ( ipsc ) - derived neural progenitor cells (NPCs) secreted proteome ( secretome separation)
[109]
P 0 of the human before multipotential induced pluripotent stem cell (iPSC) derived neural progenitor cells; put (Neural precursor cell NPC) a DMEM / basic culture medium of F-12 in N2, B-27, serum removal of Gem21 supplements added to bFGF and, Mart rijel - P coated 100 mm dishes 4 is about 90% or more cells, chilled and incubated in the dish with respect to the system. Then, the culture solution was removed, washed 2-3 times with PBS, and washed to remove the cells from the dish with Accu-rise rim enzyme with PBS once. And only the ITS was added to basic DMEM culture medium that does not contain phenol red and incubated for 24 hours on a floating state. As a control of the culture, by the ITS to the basic culture medium of the same volume was added in the same amount was used for 24 hours culture in the incubator. After collection of all of the cells in the culture medium is removed, the floating or the like by centrifugation (1000 g eseo 20 minutes) of the cell debris, and used to extract, if necessary after immediately frozen in a freezer of -70 ℃.
[110]
Cell culture conditions are as follows:
[111]
(1) Basic culture conditions of the cell - DMEM / F-12, bFGF 20 ng / ml, N2 100X- final concentration of 1X, B-27 50X- final concentration of 0.5X, 0.5X final concentration Gem21 50X-
[112]
(2) secretion proteome (secretome) obtained culture conditions for - DMEM / phenol red free, ITS 100X- final concentration of 1X
[113]
[114]
Production models of stroke
[115]
The endovascular suture insertion method (intraluminal suture method) applied to measure the neuroprotective effect on neuronal cells damage by focal cerebral ischemia were used. Zia Longa is unlike (Zea Longa, et al, Stroke., 1989, 20, 84-91) is a method in a focal cerebral ischemia model, another model has the advantage of being developed clinically similar. For this reason ischemia - a suitable model for screening mechanism research and the effects of various drugs on reperfusion (ischemia-reperfusion). Focal cerebral ischemia model has the advantage that it can observe all of the MCA (middle cerebral artery) damage which is temporarily closed by a perfusion and continuous cell damage by permanently closed using a probe in addition to the above-described advantages. Depending on the brain blood flow is determined the difference in ischemic heart (ischemic core) and the ischemic penumbra region (ischemic penumbra) E is less than 15%, and the latter is less than 40%. When closed from 10 to 20 minutes when nerve cells die sporadically causes about one hour from the heart when he animated the central part (core) and a brain injury increases, causing 2-3 hours suffered a similar injury as a permanently closed do.
[116]
After a week of acclimatization, animals were test using the breathing machwigi brought anesthesia (body weight: 250-300 g male Sprague-Dawley rat,), isoflurane (isoflurane) was used as the anesthetic. First to Rats% N 80 2 O and 20% O 2 and then the mixed gas is mixed induce general anesthesia with isoflurane at 5% was kept anesthetized with 2-2.5%. For the stroke model produced, after dissection from the left neck skin of the rat common carotid artery to the inside (common carotid artery), the outer carotid artery (external carotid artery), and then obtained by separating the internal carotid artery (internal carotid artery) of each of the blood vessel indeed, black silk tie was briefly blocked the blood flow. Cutting the carotid artery half, which was inserted into a 4-0 nylon probe (probe) of 25 ㎜ produced by rounding the cut surface through the cauterization the tip of the nylon suture to be 0.40 ㎜. After a fixed loading one nylon probe inserted through the carotid artery into the outer portion via the middle cerebral artery from the common carotid carotid insertion branched approximately 18-20 ㎜ chamber fixed to the film after the origin of the middle cerebral artery permanently close the middle cerebral artery, and then after re-suture the skin incision was natural recovery from anesthesia.
[117]
[118]
Secretion by cerebral stroke model proteome injection and behavioral experiments:
[119]
a. Human ESC- secretion proteome of injection-derived neural progenitor cells (NPC)
[120]
After establishing the stroke caused day reference point (baseline) for the behavioral experiments followed, after inserting the insulin syringe needle into the internal carotid artery region through the right outer carotid artery in a manner such as stroke model produced, 0.2 mg / kg the secretion proteome through which injection by arterial (volume 50 μ), which was administered to the culture medium or phosphate-buffered saline (PBS) in an equal volume, the control group. It was observed the state of the animals for 14 days after the injection the secretion proteome liquid, the measuring body weight and behavioral analysis was performed over a one-time infusion before and after the 4th injection.
[121]
[122]
b. Secretion proteome of human injection iPSC- derived neural progenitor cells (NPC) - multidose
[123]
After establishing the reference point (baseline) for the behavioral experiments after stroke induced day, stroke model was produced and inserted insulin syringe needle into the internal carotid artery region through the right outer carotid artery in the same manner, the secretion proteome per through which 0.7 mg / for Kg (300 g rats 200 μg, jusaryang is volume 200 μl was by intravenous (penile vein administration). control group was administered to the culture medium of the same volume. primary cultures of sheep, such as control of the secretion proteome as in proteome separation on the addition of ITS in the same amount after 24 hours of incubation the culture medium was used as control. was observed the state of the animals for 14 days after the injection the secretion proteome liquid injection before once (day 0), four times after injection It was carried out (3, 7, 10 and 14) measuring the weight and behavior analysis throughout.
[124]
[125]
c. Behavioral experiments
[126]
① body postures (torso twisting) test: to measure the asymmetrical motor behavior in order to test the upper body posture is considered as an experimental animal in the cerebral cortex and striatum sense.
[127]
[128]
The animals were evaluated a total of vestibular motor skills (gross vestibulomotor function) through the stable balance on a narrow beam: ② balance beam (beam balance) test.
[129]
[130]
③ foot fault test (foot-fault test): the test method used to test the adjustment (cooperative) and integration (synthesis) of the exercise movements (motor movement) according to the movement, foot fault (Foot-fault) is an animal does not leave the front foot and rear foot in place (misplace), and defined as the foot is dropping between the grid bars (grid bar), a foot fault is symmetrical uniquely in the normal animal.
[131]
[132]
④ capture possible traction test (Prehensile Traction test): whether acquisition of the test can was determined by the ability of animals can hang on the rope in the horizontal direction to the front foot. Capture possible traction test was performed to evaluate the muscular strength of experimental animals. The test was performed by the method reported in the prior art. A horizontal iron bar (diameter: 2 cm, length 100 cm) was placed on the 70 cm of the sponge rubber pad (7.5 cm thickness). Experimental freed after placing the paws of the animals in the iron bar. The animals were hanging on the iron bar 5 seconds. Tteoleojineunde by taking whether placing it on the iron bars of time and hind legs was calculated score as follows: 0 points - the animals are hanging on for 5 seconds, the hind legs - animals are placing on the hind legs dangling for five seconds, 1 point does not place, 2 points - experimental animals are suspended from three to four seconds, three-animals are hanging for 0-2 seconds.
[133]
[134]
⑤ open field test (open-field test): a test used to find out the common pedestrian activity level, observed the behavior patterns and characteristics of the animals was measured directly, such as the activity of the animal, sex and emotional behavior patterns.
[135]
[136]
⑥ (modified Neural Sibiarity Score) mNSS: obtained through a variety of tests of the above motor, sensory, a comprehensive record of balance, the reaction and emotional check value, was calculated according to the following criteria a score (by summing the score per individual mNSS the measurement).
[137]
- open-field test (measuring the emotional gender, activity, and behavioral patterns of animals)
[138]
No movement: 3
[139]
1-20 times: 2
[140]
21-30 times: 1
[141]
More than 30 times: 0
[142]
[143]
- acquisition possible traction tests (strength measurement)
[144]
0-5 seconds: 3
[145]
6-10 seconds: 2
[146]
11-20 seconds: 1
[147]
More than 21 seconds: 0
[148]
[149]
Beam balance test (balance measurement)
[150]
0 points: 1 = stable stance
[151]
2 = Hold the side of the beam a little shaky about
[152]
1 point: when a = 3 at least one leg from falling off the beam
[153]
4 = balanced, but if trying to catch falling
[154]
2 points: 5 = If you did not hold the balance sheet upside down on the beam run falls
[155]
6 = If no balance on the beam falling not catch
[156]
[157]
- foot fault test (motor coordination skills)
[158]
0-5 times: 0
[159]
6-10 seconds: 1
[160]
11-20 seconds: 2
[161]
More than 21 seconds: 3
[162]
[163]
- body posture test (asymmetrical motor behavior)
[164]
0: 2
[165]
1-4: 1
[166]
More than five times: 0
[167]
[168]
A rat 14 days after cerebral ischemia induced thoracotomy after anesthesia with sol retil then, after the incision Dextrocardia injecting the needle into the left ventricle by means of a pump by flowing through the phosphate buffer solution in the heart was excised after the brain tissue to remove blood . After the tissue sections for samples produced by the bregma (bregma), embedded in paraffin, and shoot the next slide is fixed behind a hedge of brain tissue sections stained with hematoxylin and dehydrated to make the damaged brain tissue with a digital camera transferred to a computer. Using an image analysis program (image J) to the stroke rate (%) was calculated by Equation 1.
[169]
Equation 1
[170]
Infarction rate (%) = (the normal area of ​​the left hemisphere-ischemic area of ​​the normal tissue in the area) / area of ​​the top left hemisphere X 100
[171]
[172]
Immunohistochemical analysis and quantification
[173]
The brain for 24 hours fixed with 4% formaldehyde and washed with PBS. For paraffin section making, and dehydrated tissue was embedded in paraffin in progressive ethanol. Paraffin were cut flower with 4 mm thick layer on a microtome the embedded brain and re in a rear progressive alcohol paraffin screen for 10 minutes in xylene. After a 10 mM citric acid in the fragment for 1 hour it was added 5% BSA solution containing PBS, 0.5% Triton X-100. Then, the brain slice ED-1 (Abcam) (1: 100), double cor tin (DCX; Abcam, 1: 100), glia fibrous acidic protein (GFAP; Millipore, 1: 100) and α- smooth muscle actin (aSMA ; Abcam, 1: and incubated at 4 ℃ for 15-17 hours with the primary antibody for 100). Primary antibodies followed by washing with, PBS overnight incubation with fluorescently labeled secondary antibody and the fragment (Alexa-Fluor®488 or 594, 1500, Molecular Probes, Eugene, OR, USA) the fragment and incubated for 1 hour It was. Using a fluorescence microscope (Olympus IX71) to obtain a fluorescence image of the fragments.
[174]
[175]
Secreted proteome analysis (Secretomics)
[176]
hESC-derived neural progenitor cells (Neural precursor cell; NPC) and the secretion proteome of human iPSC-derived neural progenitor cells secreting the proteome of a have, respectively, 4-12% gradient furnace IBEX bis-Tris was separated by SDS-PAGE gel (Invitrogen) and then a gel code blue staining reagent (Pierce) were stained gel of the protein band to appear. The stained gel is cut into 10 bands of all sizes - were carried out in a manner known to gel trypsin digestion.
[177]
In-the peptides produced by the decomposition gel it was analyzed using a Quadrupole LinearTrap (LTQ) mass spectrometer (Thermo Finnigan) in conjunction with the Nano Ultra Performance Liquid Chromatography (Eksigent Technologies). Specifically, it was applied to a trypsin-treated peptides for C18 5 micron size Regular resin is packed analytical column (75 microns X 11 cm). A 97% solution of the minute linear gradient from 45 min (0.1% formic acid in distilled water) to 60% solution B (0.1% formic acid in acetonitrile) conditions of 0.3 micro-liter / hour flow rate from was carried out. The isolated peptide ions were sprayed with electric nano Electrospray Ionization (ESI) source. MS / MS spectra were the 5 most spectrum to screen the full MS scan fragments results were obtained by scanning dependent. A repetition factor for a dynamic rule to 1, the repeating period of 30 seconds, 180 seconds, a dynamic exclusion duration, 1.5 Da mass exclusion width, and sets the operation exclusion list to 50.
[178]
Peptides and proteins was confirmed by using the search algorithm turbo -SEQUEST (Thermo Finnigan) at ipi.HUMAN v3.76 database (89 378 entries). After the database search was confirmed that the peptides and proteins identified using the scarf fold 2 (Proteome Software). It was selected for a set of peptides having 0.95 or more peptides peuropet (PeptideProphet) of the peptide obtained by the probability SEQUEST search. In addition, the protein was obtained with a list of at least 0.99 peuropet protein (ProteinProphet) and the probability of two or more specific peptide.
[179]
[180]
Spinal cord injuries (Spinal cord injury: SCI) model making, minutes Katowice injection and behavioral recovery test (BBB score test)
[181]
Experimental animals were subjected to vertebrae removed after anesthesia (body weight: 250-300 g male Sprague-Dawley rat,) (laminectomy). Tteuryeoseo using the NYU Impactor fall over the spinal cord exposed using a 10 g old rod was induced spinal cord injury (SCI model making). Since then a well disinfected the wound was sutured to the skin. After receiving the spinal cord around the multipotential stem cells (ESC) of the present invention in the wound part derived neural progenitor cells; was injected (injection) the secretion proteome (secretome) of (Neural precursor cell NPC). After an interval of about three days was administered intravenously to two more times. After injection the secretion proteome (secretome) BBB test was performed weekly. By the BBB test conducted over 8 weeks compared to the control group.
[182]
[183]
Statistical Analysis
[184]
Statistical significance between groups was determined by using a one-wat analysis of variance (ANOVA) Tukey's correction was applied, p value <0.05 was considered significant statistically.
[185]
[186]
1 result: human embryonic stem cells (ESC) - the secreted proteome-derived neural progenitor cells
[187]
In the stroke model, human ESC- derived neural progenitor cells; to see the improvement of the disease caused by the secretion proteome of (Neural precursor cell NPC) was carried out for two weeks for the next three tests of the group. After stroke induced 24 hours, and randomly assigned to a disease is identified rat induced in the behavioral experiments with three groups secretion Next, the right outer carotid proteome (0.2 mg / kg, volume 50 ㎕), the same volume of the medium or PBS (50 ㎕) was injected. After each injection the material determine the condition and body weight of the animal to 3, 7, 10 and 14, which was subjected to behavioral analysis.
[188]
TABLE 1
group Explanation
PBS control PBS-treated group after stroke induced
Media control After a stroke caused by media treatment groups used in neural progenitor cell culture
Secretion proteome treated group After stroke induced neural progenitor cell culture-treated group

[189]
[190]
[191]
Results ischemic lesion analysis
[192]
Induction of stroke with permanent MCAO in rats induced damage to the extensive brain lesions. By extraction of the brain causing a stroke after a lapse of 14 days following, TTC (2,3,5-triphenyltetrazolium chloride) was confirmed brain damage and whether damage to the stained area. TTC staining is there is stained with normal mitochondrial oxidative enzyme system (mitochondrial oxidative enzyme system) and the reaction in the cell, when mitochondrial damage accept ischemic damage oxidation system is disturbed staining does not exhibits a white to distinguish the damaged area of ​​the brain can.
[193]
As shown in Fig. 1, the damage caused by middle cerebral artery occlusion was mainly generated in the cortex and the corpus striatum region of the right brain (Fig. 1). In addition, the secretion proteome injection of neural precursor cells reduced the ischemic lesion (infarct size). PBS control was close to 60% damage to the right brain, the medium control group but 46% of the damage, the secretion proteome-treated group was significantly reduced compared to the control is damaged, so the damage site 29%.
[194]
[195]
Results Weight Analysis
[196]
Stroke causes the proceeds immediately to reduce the weight of seven days to reduce the exercise capacity of rats, a variety of drugs leads to an increase in body weight over the recovery of the athleticism. Similar to the cell transplant infusion of secretion proteome to induce weight gain (Fig. 2). Symptoms The most prominent in the recovery of the nerve damage is the restoration of body weight. Secretion proteome treated group showed significant improvement compared to the PBS control.
[197]
[198]
Results Behavioral Analysis
[199]
Secretion proteome treated group showed a statistically significant improvement in behavior in comparison to the effect of both the control group in the beam balance test, the effect was an immediate effect such as appearing from the treatment day 3 (Fig. 3a).
[200]
In addition, the acquisition can be treated in the traction test (prehensile traction) (injection) showed significant effect in both the control group compared statistically to day 7 (Fig. 3b).
[201]
Moreover, the secretion proteome injection showed the effect of reducing the offense (foot fault) frequency in the net (Fig. 3c). Units of measurement line that's vigorous actions per hour showed that the trend also improved cross (line cross) (Fig. 3d).
[202]
[203]
Improvement (mNSS) analysis Comprehensive neurological behavior
[204]
(Modified Neurological Severity Score test) mNSS is a scheme for measuring the neurological function. Movement (muscle tone) with mensory (visual, tactile, and proprioception (proprioceptive)) were evaluated by the entry. Normal is zero, it is determined that the higher the score, the more severe features. As shown in FIG 4, the secretion proteome analysis of the treatment groups in the mNSS showed a much higher therapeutic effect (improvement action) than the PBS control medium and the control group in the early treatment (Figure 4).
[205]
PBS control group in the mNSS test is an average of 5.5 points 14 days in average of 5.4 points after 1 day induced a cerebral ischemia, I could see the neurobehavioural disorders maintained by stroke. For the medium control group and showed a transient behavior improvement effect of treatment 3 days, it did not exert an additional effect of improving. On the other hand, in the case of the secretion proteome is treated group showed a constant behavior improvement from treatment 3 days (mNSS 4.5 points) to 10 (mNSS 3 points) effect.
[206]
[207]
Secreted proteome analysis
[208]
iPSCs derived neural progenitor cells; secretion obtained from (Neural precursor cell NPC) proteomic was includes the following proteins: Ah drawn (Agrin), Annexin A5 (Annexin A5), BSG (Basigin), bigeulri compartment (Biglycan) , Carl ponin -3 (Calponin-3), Cocteau Shin-like proteins (Coactosin-like protein), nose Lin -1 (Cofilin-1), collagen alpha-2, tuberculin -3 (Cullin-3), des trim ( Destrin), a distributor glycans (Dystroglycan), F. Lin -B2 (Ephrin-B2), Expo tin -2 (Exportin-2), Lin Eze (Ezrin), fibronectin, blood Dublin -1 (Fibulin-1), Frizzled -related protein, gelatin-binding protein -3 (Galectin-3 binding protein), granules Lin (Granulins), growth / differentiation factor 11 (Growh / differentiation factor 11), haptoglobin (haptoglobin), hepo peksin (Hemopexin), High mobility group protein B2, No. nerin (Hornerin), imported tin -9 (Importin-9), insulin-like growth factor binding protein 2 (insulin-like grwoth factor-binding protein 2), lupus La protein (lupus La protein), the macrophages Copper inhibitor (Macrophage migration inhibitory factor), mid-Keen (Midkine), Moe Shin (Moesin), Neuro pilrin 2 (Neuropilin 2), the play Upper pin (Pleiotrophin), propyl Lin -1 (Profilin-1), Protein DJ -1 (Protein DJ-1),
[209]
Neural progenitor cells of the human embryonic stem cells; secreted proteome obtained from (Neural precursor cell NPC) was to include the following proteins: Ahh Green (Agrin), annexin A2 (Annexin A2), art raktin (Attractin), bigeulri Khan (Biglycan), serul to plasmin (Ceruloplasmin), nose Lin -1 (Cofilin-1), collagen alpha-1, co Ronin -1X (Coronin-1X), when the dummy Dean (Dermicidin), DERP12, F. Lin -B3 , cytosine exo -2 (Exostosin-2), Lin Eze (Ezrin), gel Larkin -3-binding protein, granules Lin (Granulins), growth / differentiation factor 11 (Growh / differentiation factor 11), haptoglobin (haptoglobin), hemo peksin (Hemopexin), High mobility group protein B2, No. nerin (Hornerin), insulin-like growth factor binding protein 2 (insulin-like grwoth factor-binding protein 2), number of loops La protein, mid Keen (Midkine), Moe new (Moesin), multiple epidermal growth factor-like domains protein 8 (multiple epidermal growth factor-like domains protein 8), you Dogen -1 (Nidogen-1), para thymosin (para thymosin), propyl Lin -2 (Profilin-2), protein DJ-1 (Protein DJ-1), Secreted frizzled-related protein-2, SAT sikeuri Yes Nin (Secretogranin), Tallinn -1 (Talin-1), T thymosin beta -4 (Thymosin beta-4),
[210]
[211]
Spinal cord injury therapy
[212]
5 places into injecting secreted proteome (secretome) about 30 μl of; (NPC Neural precursor cell) (: (SCI Spinal cord injury) to create a model directly before multipotent stem cells (ESC) derived neural progenitor cells in the damaged part of the spinal cord injury It was injection). Then the double-secretion proteome more vein with an interval about 3 days and injected by about 200 μg (200 μl). Every action was evaluated according to a spinal cord injury recovery test method (BBB test) with respect to the recovery act. BBB Test is a well-known test method typically for spinal cord injury research. BBB test carried out at least 8 weeks, showed an excellent recovery behavior results in secretion proteome treated group (injection) as compared to the untreated control group (control) (Fig. 12).
[213]
[214]
Experimental Result 2: The human induced pluripotent stem cell (iPSC) - secretion proteome + multidose derived neural progenitor cells
[215]
Neural precursor cells in a stroke model; to see the improvement of the disease by the administration of a single dose and multidose (Neural precursor cell NPC) secretion proteome was carried out for two weeks following the experiment with 4 groups of. Experimental schedule is shown in Figure 5.
[216]
TABLE 2
group Explanation
Controls Untreated group after stroke induced
Media control After a stroke caused by media treatment groups used in neural progenitor cell culture
Secretion proteome per treatment group After stroke induced neural progenitor cell culture one time treated group
Secretion proteome fourth group treated After stroke induced neural progenitor cell culture fourth group treated

[217]
[218]
[219]
Figure 6a to 6i are multipotential before iPS cells derived from-a single dose of the neural progenitor cells secreting proteomic vs. 4th indicates the result of the administration. The secretion proteome (secretome-M) for 4 times in the group almost all conditions, it can be seen that demonstrate the superior behavior improvement compared to the other groups. That is, it was confirmed that the multidose group exerts a very significant therapeutic effects in improved behavior compared to the single administration group.
[220]
[221]
Results Weight Analysis
[222]
Secretion proteome single dose and multidose treatment group showed a significant improvement in the treatment Day 3 compared to the PBS control group (Fig. 6i). However, the weight differences between groups, as the elapsed time is lost the statistical significance.
[223]
[224]
Results Behavioral Analysis
[225]
Body posture (torso twisting) secretion proteome multidose treatment groups in the inspection to induce statistically significant brain lesions in the left side of the body to improve the asymmetric movement effects than the other groups (Fig. 6b). Whereas intact jjokin right asymmetric body movement (Fig. 6c) of the groups were not statistically significant and was observed except for the 10 days of the secreted proteome tea party groups.
[226]
Secretion proteome repeated administration stumble on the net (foot fault) eoteuna demonstrate a tendency to reduce the frequency, and the control group and was not statistically significant except for 14 (Fig. 6d). Line unit for measuring the time that the active behavior per cross (cross line) also showed a trend of improvement in single dose and multidose the secretion proteome (Fig. 6e).
[227]
Rearing scores also were the single and repeated administration of the secretion proteome determine the tendency to lead to improvements as compared to the other experimental groups (Fig. 6f), improve by the multidose the secretion proteome statistically significant compared to the two control action in the beam balance test effect the showed, the effect was an immediate effect such as appearing from the treatment day 3 (Fig. 6g). In addition, the acquisition can be treated in the traction test (prehensile traction) (injection) showed significant effect in both the control group compared statistically to day 7 (Fig. 6h).
[228]
[229]
Improvement (mNSS) analysis Comprehensive neurological behavior
[230]
As shown in Fig. 6a, secretion proteome multidose treatment groups in the analysis of the mNSS showed a much higher therapeutic effect (improvement action) than the PBS control medium and the control group in the early treatment. Secretion proteome was exert a significant therapeutic effect for the duration of the experiment as compared with single treatment Islands control group showed a superior action compared to the medium to improve the control effect.
[231]
[232]
Results ischemic lesion analysis
[233]
7 is a neural progenitor cells in the rat model pMCAo (Neural precursor cell; NPC) shows the effect on infarct size when administered 14 days elapsed derived secretion proteome. PBS (control), medium control (blank), NPC- secretion derived secretion proteome single (Secretome-S) or proteomic derived NPC- repeated administration (Secretome-M) treatment in the rat shows a representative image after 14 days. When administered 14 days has elapsed in the secretion of neural progenitor cells derived from a rat model pMCAo proteome shows the effect on infarct size. Upon administration of a single dose and multidose * P <0.01, as compared to both the PBS group. In particular, it was confirmed a significant improvement of P <0.05 compared to the multidose during the medium control group (blank) of the secretion proteome.
[234]
[235]
Anti-inflammatory effects - ED-1-positive cells reduced
[236]
Figure 8a-8b show that the frequency of the ipsilateral striatum of ED-1 positive cells reduced by treatment of the resulting NPC- secretion proteome, and the repeated administration of the secretion proteome causing a reduction in ED-1 positive cells significantly. Scale bar: 200 μm. ED-1 positive cells was determined the number of at least five distinct regions microscope. Measurements are expressed as ± standard error. A comparison * P <0.001 between repeated administration group - Control and secretion proteome.
[237]
[238]
Anti-inflammatory effects - reduced GFAP-positive cells
[239]
Figure 9a-9b show that reduces the incidence of ipsilateral striatum of GFAP-positive cells in the treatment of NPC- derived secretion proteome, the repeated administration of the secretion proteome significantly induced a decrease in the GFAP-positive cells. In particular, the expression of GFAP induced the secretion proteome repeated significantly decreased in the treatment group than in the control group the medium (Blank). Scale bar: 200 μm. The number of GFAP-positive cells was determined in at least five separate areas of a microscope. Measurements are expressed as ± standard error. When multiple comparisons between each group * P <0.05 and *** P <0.001
[240]
[241]
Operation effects of the intrinsic stem cells
[242]
10 shows the operation of the endogenous neural stem cells (endogenous neural stem cell) in rat brain tissue caused by the administration of the secretion proteome. Secretion proteome of administration was tested after 14 days. DCX (red) positive cells were observed in the rat brain ischemia. Scale bar: 500 μm.
[243]
[244]
Increased angiogenic effect
[245]
11 shows a significantly higher frequency of the repeated administration of the ipsilateral striatum α-SMA positive vessels in the treatment of increased NPC- derived secretion proteome and secretion proteome increasing the number of blood vessels. The number of α-SMA positive vessels were measured at least five distinct regions microscope. Measurements are expressed as ± standard error. When a comparison of the repeat dose (Secretome-M) group, * P <0.005 - Control and secretion proteome.
[246]
[247]
Having described a preferred embodiment of the present invention, these specific techniques according to those skilled in the art, is just an example only a preferred embodiment, it is obvious that this is not to be limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by appended claims and their equivalents.
[248]

Claims

[Claim 1]Neural progenitor cells (neural precursor cell; NPC) ischemic diseases or neurological inflammatory disease therapeutic composition comprising a secretion proteome (secretome) as an active ingredient.
[Claim 2]
The method of claim 1, wherein the neural precursor cell composition, characterized in that the differentiation from multipotent before pluripotent stem cells.
[Claim 3]
The method of claim 1, wherein the neural precursor cell composition, characterized in that the neural rosette stage before and after the neural progenitor cells differentiated from pluripotent stem cells, multipotent ago.
[Claim 4]
The method of claim 2 or claim 3 wherein the former multipotential pluripotent stem cells are embryonic stem cells (embryonic stem cells), iPSCs (induced pluripotent stem cells), embryonic germ cells (embryonic germ cells) or embryonic tumor cells (embryonic carcinoma cells ) the composition characterized in that.
[Claim 5]
The method of claim 1, wherein the neural progenitor cells are PSA-NCAM (poly-sialylated neural cell adhesion molecule) - the composition, characterized in that the positive neural progenitor cell.
[Claim 6]
The method of claim 1, wherein the neural progenitor cells are PSA-NCAM (poly-sialylated neural cell adhesion molecule) - a composition which is characterized in that the voice neural progenitor cells.
[Claim 7]
The method of claim 1 wherein the secretion proteome composition is to be contained in the cell culture medium obtained by culturing the neural precursor cells in cell culture medium to form the features.
[Claim 8]
The method of claim 7, wherein the cell culture medium is serum-free containing a neural progenitor cell ITS (basic fibroblast growth factor) (insulin / transferrin / selenium) and bFGF cell being obtained by removing the cells were cultured in a culture medium the composition.
[Claim 9]
The method of claim 1 wherein the secretion proteome is a composition comprising a protein of the following: O green (Agrin), Annexin A5 (Annexin A5), BSG (Basigin), bigeulri compartment (Biglycan), knife ponin - 3 (Calponin-3), Cocteau Shin-like proteins (Coactosin-like protein), nose Lin -1 (Cofilin-1), collagen alpha-2, tuberculin -3 (Cullin-3), des trim (Destrin), Distributor a glycan (Dystroglycan), F. Lin -B2 (Ephrin-B2), Expo tin -2 (Exportin-2), Lin Eze (Ezrin), fibronectin, blood Dublin -1 (Fibulin-1), Frizzled-related protein, gelatin -3-binding protein (Galectin-3 binding protein), granules Lin (Granulins), growth / differentiation factor 11 (Growh / differentiation factor 11), haptoglobin (haptoglobin), hepo peksin (Hemopexin), High mobility group protein B2 , No. nerin (Hornerin), imported tin -9 (Importin-9), insulin-like growth factor binding protein 2 (insulin-like grwoth factor-binding protein 2), lupus La protein (lupus La protein), macrophage migration inhibitory factor (Macr ophage migration inhibitory factor), mid-Keen (Midkine), Moe Shin (Moesin), Neuro pilrin 2 (Neuropilin 2), the play Upper pin (Pleiotrophin), propyl Lin -1 (Profilin-1), protein DJ-1 (Protein DJ-1), La diksin (Radixin),
[Claim 10]
The method of claim 1, wherein the composition comprising the proteins of the secretion proteome is to: a ah green (Agrin), Annexin A2 (Annexin A2), art raktin (Attractin), bigeulri compartment (Biglycan), serul plasmin (Ceruloplasmin), nose Lin -1 (Cofilin-1), collagen alpha-1, co Ronin -1X (Coronin-1X), when the dummy Dean (Dermicidin), DERP12, F. Lin -B3, exo-cytosine-2 ( Exostosin-2), Lin Eze (Ezrin), gel Larkin -3-binding protein, granules Lin (Granulins), growth / differentiation factor 11 (Growh / differentiation factor 11), haptoglobin (haptoglobin), hemo peksin (Hemopexin), High mobility group protein B2, No. nerin (Hornerin), insulin-like growth factor binding protein 2 (insulin-like grwoth factor-binding protein 2), the loop number La protein, mid Keen (Midkine), Moe Shin (Moesin), multiple epidermal growth factor-like domain protein 8 (Multiple epidermal growth factor-like domains protein 8), you Togendai -1 (Nidogen-1), p-thymosin (Parathymosin), propyl Lin -2 (Profilin- 2) the protein DJ-1 (Protein DJ-1), Secreted frizzled-related protein-2, sikeuri Sat Yes Nin (Secretogranin), Tallinn -1 (Talin-1), thymosin beta -4 (Thymosin beta-4) , TGFBI (Transforming grwowth factor-beta-induced protein ig-h3),
[Claim 11]
The method of claim 1, wherein the ischemic disease is a composition, wherein is selected from ischemic cerebrovascular disease, ischemic heart disease, myocardial infarction, angina pectoris, ischemic heart disease, limb end the group consisting of ischemic disease not.
[Claim 12]
The method of claim 11, wherein said ischemic cerebrovascular disease is a composition wherein the ischemic stroke.
[Claim 13]
The method of claim 1 wherein the nerve inflammation disease is the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, diffuse Lewy body disease, white matter encephalitis, temporal lobe epilepsy, and spinal cord injury composition, wherein is selected from the.
[Claim 14]
The method of claim 1, wherein the ischemic disease or a composition for treating neurological inflammatory disease is a composition, characterized in that one-time administration of tolerated.
[Claim 15]
The method of claim 1, wherein the ischemic disease or a composition for treating neurological inflammatory disease is a composition wherein the multidose tolerated.
[Claim 16]
(A) a neural precursor cell (neural precursor cell; NPC) a therapeutically effective amount of a proteome derived secretion; And (b) a composition comprising a carrier (carrier) that is pharmaceutically acceptable; ischemic disease or a method for prevention or treatment of neurological inflammatory disease, comprising administering to a subject (subject) for.