Treatment of amyotrophic lateral sclerosis with extracellular vesicle compositions

Abstract

A method for treating amyotrophic lateral sclerosis by administering a therapeutic MSC secretome product prepared by a method comprising the step of culturing bone marrow-derived MSCs under conditions including an oxygen pressure of less than 5% and a culture medium with a pH of less than 7 is disclosed.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 507,204, filed on June 9, 2023, and U.S. Provisional Patent Application No. 63 / 570,632, filed on March 27, 2024, which are hereby incorporated by reference in their entirety.

Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a devastating terminal neurodegenerative disease that affects approximately 4.5 people per 100,000. ALS is the most common type of motor neuron disease and the third most common neurodegenerative disease after Alzheimer's disease and Parkinson's disease. ALS is a nervous system disease that affects the brain and spinal cord, causing loss of muscle control. Approximately 4.5 people per 100,000 live with this disease, but unfortunately, there has been little progress in the understanding and treatment of ALS. Current diagnosis is one of exclusion based on symptoms. Currently, there is no treatment for ALS, and the disease worsens over time. Unfortunately, there is no medical treatment to stop or reverse the progression of ALS, and access to the aforementioned treatments may be limited and restricted. There is a need for a safe and effective treatment for ALS.

Summary of the Invention

[0003] In some aspects herein, a method of treating ALS in a subject that requires treatment for ALS, the method comprising administering to the subject a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition comprising extracellular vesicles, wherein at least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 CD9 - CD81 - is disclosed.

[0004] In some embodiments, the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score of the subject increases by at least about 0.1 point per month after administration as compared to the ALSFRS-R score measured before administration, or the ALSFRS-R score decreases by less than about 3.0 points per month. In some embodiments, the ALSFRS-R score of the subject increases by at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration as compared to the ALSFRS-R score measured before administration. In some embodiments, the ALSFRS-R score of the subject decreases by less than about 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration as compared to the ALSFRS-R score measured before administration. In some embodiments, the subject has a history of the ALSFRS-R score decreasing by about 3.0 points per month before administration of the therapeutic MSC secretome composition.

[0005] In some aspects herein, a method of treating amyotrophic lateral sclerosis (ALS) in a subject who needs treatment for ALS, the method comprising administering to the subject a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition comprising extracellular vesicles, wherein the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score of the subject increases by at least about 0.1 point per month after administration as compared to the ALSFRS-R score measured before administration, or the ALSFRS-R score decreases by less than about 3.0 points per month, is disclosed.

[0006] In some embodiments, the target ALSFRS-R score increases by at least approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subject's ALSFRS-R score decreases by approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subject has a history of a monthly decrease of approximately 3.0 points in their ALSFRS-R score prior to administration of the therapeutic MSC secretome composition.

[0007] In some embodiments, at least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 + CD9 - CD81 -In some embodiments, the therapeutic MSC secretome composition includes the following proteins: ferritin, NUP85, LAMP2, GPR115, serpin Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF R-alpha, soltirin, serpin B6, Dkk-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20 R-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecane-4 (one or more), as well as: Ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-IRA, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirre13, FLRT1, Galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurograinin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLIN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a SDF-lb, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.1, EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-Gamma-R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein-11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 It contains at least one protein selected from Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, or IL-13.

[0008] In some embodiments, the therapeutic MSC secretome composition comprises the following nucleic acids: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1’7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-19’7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p,hsa-miR-26a-5p, hsa-miR-27a-3p, hsa-mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR -29c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-mir-30d , hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR- 320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3 p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa- mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-66 Includes one or more of the following: 3a, hsa-miR-874-3p, hsa-mir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, or hsa-miR-99b-5p.

[0009] In some embodiments, the composition is produced by the steps of (a) culturing bone marrow-derived MSCs in a culture medium with a pH of less than 7 under the following conditions: (i) an oxygen tension of less than 5% and (ii) a pH of less than 7 to produce an MSC-conditioned medium; (b) collecting the MSC-conditioned medium; and (c) formulating the MSC-conditioned medium to produce a therapeutic MSC secretome composition, wherein the therapeutic MSC secretome composition contains proteins and extracellular vesicles produced by the bone marrow-derived MSCs in step (a). In some embodiments, the culture medium is serum-free. In some embodiments, the glucose concentration of the culture medium is less than 4.5 g / L.

[0010] In some embodiments, the subject has spinal onset ALS. In some embodiments, the subject has bulbar onset ALS. In some embodiments, the subject has progressive ALS. In some embodiments, the subject exhibits limb-related symptoms. In some embodiments, the subject exhibits dysphagia or dysarthria. In some embodiments, treatment slows the progression of ALS.

[0011] In some embodiments, the subject has one or more amino acid mutations in the SOD1 protein. In some embodiments, one or more amino acid mutations include G93A. In some embodiments, the subject has one or more dipeptide repeats in the C9ORF72 protein. In some embodiments, one or more dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

[0012] In some embodiments, the subject is human. In some embodiments, the bone marrow-derived MSCs are derived from human bone marrow.

[0013] In some embodiments, the step of administering includes intravenous administration. In some embodiments, the dosage of the therapeutic MSC secretome composition administered to the subject is a cell equivalent dosage of 0.7 to 7 million cells per kg. In some embodiments, the therapeutic MSC secretome composition contains 4×10 10 ~10×10 10 cells per ml. In some embodiments, the therapeutic MSC secretome composition contains 5×10 11 ~1.5×10 12 extracellular vesicles. In some embodiments, the composition is administered monthly over a period of 2 months or more, or once every 1 month, 2 months, or 3 months or more.

[0014] In some aspects of this specification, a method is disclosed for producing a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition for treating ALS in subjects requiring treatment for amyotrophic lateral sclerosis (ALS), comprising the steps of: (a) culturing bone marrow-derived MSCs in a culture medium under the following conditions: (i) an oxygen tension of less than 5% and (ii) a pH of less than 7 to produce an MSC-conditioned medium; (b) collecting the MSC-conditioned medium; and (c) formulating the MSC-conditioned medium to produce a therapeutic MSC secretome composition, wherein the therapeutic MSC secretome composition comprises proteins and extracellular vesicles produced by the bone marrow-derived MSCs in step (a).

[0015] In some embodiments, the culture medium is serum-free. In some embodiments, the glucose concentration of the culture medium is less than 4.5 g / L. In some embodiments, at least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 + CD9 - CD81 - In some embodiments, bone marrow-derived MSCs are derived from human bone marrow.

[0016] In some embodiments, the therapeutic MSC secretome composition includes the following proteins: ferritin, NUP85, LAMP2, GPR115, serpin Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF R-alpha, soltirin, serpin B6, Dkk-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20 R-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecane-4 (one or more), as well as: Ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-IRA, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirre13, FLRT1, Galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurograinin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLIN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 Alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg 4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.1, EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-Gamma-R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein 11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecane-4, IL-2 Further comprising at least one protein selected from Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, or IL-13.

[0017] In some embodiments, the extracellular vesicles contain the following nucleic acids: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1’7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-19’7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p,hsa-miR-26a-5p, hsa-miR-27a-3p, hsa-mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR -29c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-mir-30d , hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR- 320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3 p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa- mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-66 Includes one or more of the following: 3a, hsa-miR-874-3p, hsa-mir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, or hsa-miR-99b-5p.

[0018] In certain aspects of this specification, the use of compositions produced by any of the methods described herein in the treatment of amyotrophic lateral sclerosis (ALS) in subjects requiring treatment for ALS is disclosed.

[0019] In some embodiments, the subject has spinal-onset ALS. In some embodiments, the subject has medullary-onset ALS. In some embodiments, the subject has progressive ALS. In some embodiments, the subject exhibits limb-related symptoms. In some embodiments, the subject exhibits dysphagia or difficulty speaking. In some embodiments, the progression of ALS is slowed by treatment.

[0020] In some embodiments, the subject has one or more amino acid mutations in the SOD1 protein. In some embodiments, the one or more amino acid mutations include G93A. In some embodiments, the subject has one or more dipeptide repeats in the C9ORF72 protein. In some embodiments, the one or more dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

[0021] In some embodiments, the subject is human. In some embodiments, the composition is administered intravenously to the subject.

[0022] In some embodiments, the ALS Functional Rating Scale-R (ALSFRS-R) score of the subject increases by at least approximately 0.1 points per month after administration compared to the ALSFRS-R score measured before administration, or the ALSFRS-R score decreases by less than approximately 3.0 points per month. In some embodiments, the ALSFRS-R score of the subject increases by at least approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration compared to the ALSFRS-R score measured before administration. In some embodiments, the subject's ALSFRS-R score decreases by approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subject has a history of a monthly decrease of approximately 3.0 points in their ALSFRS-R score prior to administration of the therapeutic MSC secretome composition.

[0023] In some embodiments, the dose of the therapeutic MSC secretome composition administered to the subject is a cellular equivalent dose of 0.7 to 7 million cells per kg. In some embodiments, the therapeutic MSC secretome composition is 4 × 10 per ml. 10 ~10×10 10 Contains 5 × 10 cells. In some embodiments, the therapeutic MSC secretome composition contains 5 × 10 11 ~1.5×10 12 It contains 1 extracellular vesicle. In some embodiments, the composition is administered monthly for a period of two months or more, or once every one, two, or three months or more.

[0024] In some embodiments herein, the use of a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles in the treatment of amyotrophic lateral sclerosis (ALS) in subjects requiring treatment for ALS, wherein at least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 + CD9 - CD81 - The use is disclosed. In some embodiments, the composition is administered intravenously to the subject.

[0025] In some embodiments, the ALS Functional Rating Scale-R (ALSFRS-R) score of the subject increases by at least approximately 0.1 points per month after administration compared to the ALSFRS-R score measured before administration, or the ALSFRS-R score decreases by less than approximately 3.0 points per month. In some embodiments, the ALSFRS-R score of the subject increases by at least approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration compared to the ALSFRS-R score measured before administration. In some embodiments, the subject's ALSFRS-R score decreases by approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subject has a history of a monthly decrease of approximately 3.0 points in their ALSFRS-R score prior to administration of the therapeutic MSC secretome composition.

[0026] In certain aspects of this specification, the use of a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles in the treatment of amyotrophic lateral sclerosis (ALS) in subjects requiring treatment for ALS is disclosed, wherein the subject's ALS Function Rating Scale-R (ALSFRS-R) score increases by at least approximately 0.1 points per month after administration, or decreases by less than approximately 3.0 points per month, compared to the ALSFRS-R score measured before administration.

[0027] In some embodiments, the target ALSFRS-R score increases by at least approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subject's ALSFRS-R score decreases by approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subject has a history of a monthly decrease of approximately 3.0 points in their ALSFRS-R score prior to administration of the therapeutic MSC secretome composition.

[0028] In some embodiments, at least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 + CD9 - CD81 -In some embodiments, the therapeutic MSC secretome composition includes the following proteins: ferritin, NUP85, LAMP2, GPR115, serpin Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF R-alpha, soltirin, serpin B6, Dkk-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20 R-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecane-4 (one or more), as well as: Ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-IRA, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirre13, FLRT1, Galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurograinin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLIN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a SDF-lb, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.1, EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-Gamma-R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein-11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 Further comprising at least one protein selected from Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, or IL-13.

[0029] In some embodiments, the extracellular vesicles contain the following nucleic acids: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1’7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-19’7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p,hsa-miR-26a-5p, hsa-miR-27a-3p, hsa-mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR -29c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-mir-30d , hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR- 320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3 p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa- mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-66 Includes one or more of the following: 3a, hsa-miR-874-3p, hsa-mir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, or hsa-miR-99b-5p.

[0030] In some embodiments, the composition is produced by the steps of (a) culturing bone marrow-derived MSCs in a culture medium with a pH of less than 7 under the following conditions: (i) an oxygen tension of less than 5% and (ii) a pH of less than 7 to produce an MSC-conditioned medium; (b) collecting the MSC-conditioned medium; and (c) formulating the MSC-conditioned medium to produce a therapeutic MSC secretome composition, wherein the therapeutic MSC secretome composition comprises proteins and extracellular vesicles produced by the bone marrow-derived MSCs in step (a).

[0031] In some embodiments, the culture medium is serum-free. In some embodiments, the glucose concentration of the culture medium is less than 4.5 g / L.

[0032] In some embodiments, the subject has spinal-onset ALS. In some embodiments, the subject has medullary-onset ALS. In some embodiments, the subject has progressive ALS. In some embodiments, the subject exhibits limb-related symptoms. In some embodiments, the subject exhibits dysphagia or difficulty speaking. In some embodiments, the progression of ALS is slowed by treatment.

[0033] In some embodiments, the subject has one or more amino acid mutations in the SOD1 protein. In some embodiments, the one or more amino acid mutations include G93A. In some embodiments, the subject has one or more dipeptide repeats in the C9ORF72 protein. In some embodiments, the one or more dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

[0034] In some embodiments, the subject is human. In some embodiments, bone marrow-derived MSCs are derived from human bone marrow.

[0035] In some embodiments, the composition is administered intravenously to the subject. In some embodiments, the dose of the therapeutic MSC secretome composition administered to the subject is a cellular equivalent dose of 0.7 to 7 million cells per kg. In some embodiments, the therapeutic MSC secretome composition is 4 × 10⁶ per ml. 10 ~10×10 10 Contains 5 × 10 cells. In some embodiments, the therapeutic MSC secretome composition contains 5 × 10 11 ~1.5×10 12 It contains 1 extracellular vesicle. In some embodiments, the composition is administered monthly for a period of two months or more, or once every one, two, or three months or more.

[0036] Reference The patents, publications, and non-patent documents cited herein are each incorporated herein by reference in whole, as if each were each incorporated by reference individually. If any publication or patent or patent application incorporated by reference conflicts with any disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material. [Brief explanation of the drawing]

[0037] The features of this disclosure are specifically stated in the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description, which specifies exemplary embodiments in which the principles of this disclosure are utilized, and to the appended drawings (also referred to herein as "Figure" and "FIG.").

[0038] [Figure 1A] This figure shows the difference in raw ALSFRS-R scores across all subjects during each measurement period. [Figure 1B] This figure shows fitted linear regression analysis over time for each subject to illustrate disease progression during the trial. [Modes for carrying out the invention]

[0039] I. Definition As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise specified. Also note that the term “or” is generally used to mean “and / or” unless otherwise specified. As used herein, the terms “and / or,” “any combination thereof,” and their grammatical equivalents are interchangeable. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C. The term “or” may be used conjunctively or disjunctively unless the context specifically refers to a disjunctive use.

[0040] The terms “about” or “approximately” can mean within an acceptable margin of error for a particular value, which may depend in part on how that value is measured or determined, for example, on the limitations of the measurement system. For example, “about” could mean within or above one standard deviation. Alternatively, “about” could mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term could mean being within one order of magnitude, up to five times, or up to two times the value. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be assumed to mean within an acceptable margin of error for that particular value.

[0041] Throughout this disclosure, numerical features are presented in range form. It should be understood that this range form is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of all embodiments. Therefore, unless otherwise explicitly indicated in the context, range descriptions should be interpreted as disclosing possible subranges as well as individual numerical values ​​within that range to a lower limit of one-tenth of a unit. For example, a range description such as 1–6 should be considered to disclose not only subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, as well as individual values ​​within that range, e.g., 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the width of the range. These intervening upper and lower limits may independently be included within smaller ranges and, furthermore, are subject to any specifically excluded limitations within the ranges contained and expressed in this disclosure. If a specified range includes one or both of the limit values, the range excluding one or both of those limit values ​​is also included in this disclosure unless the context explicitly indicates otherwise.

[0042] As used herein and in the claims, the words “comprising” (and any form such as “comprise” or “comprises”), “having” (and any form such as “have” or “has”), “including” (and any form such as “includes” or “include”), or “containing” (and any form such as “contains” or “contain”) are inclusive or open-ended and do not preclude any additional elements or method steps not enumerated. All embodiments discussed herein can be implemented with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the compositions of the Disclosure can be used to achieve the methods of the Disclosure.

[0043] References to “some embodiments,” “an embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described in relation to that embodiment are included in at least some embodiments of this disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0044] To provide a complete understanding of the various embodiments, certain specific details are described herein. However, those skilled in the art will understand that this disclosure can be implemented without these details. In other instances, well-known techniques or methods are neither shown nor described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless otherwise required in context, throughout this specification and the following claims, the word “comprise,” and variations such as “comprises” and “comprising,” should be interpreted as having an open and comprehensive meaning, i.e., “including, but not limited.” Furthermore, the headings provided herein are for convenience only and do not imply any interpretation of the scope or meaning of the claimed embodiments.

[0045] Similar or equivalent methods and materials described herein may be used in the implementation or testing of this disclosure, although preferred methods and materials are described below.

[0046] II. Therapeutic composition In addition to the components used to prepare the compositions disclosed herein, the compositions themselves used in the methods disclosed herein are disclosed. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to each of the various individual combinations, collective combinations, and permutations of these compounds may not be expressly disclosed, and should be understood that each is specifically intended and described herein. For example, where a particular MSC secretome (including, but not limited to, MSC exosomes (with or without growth factors) referred herein as extracellular vesicle isolation products (EVIPs)) is disclosed and discussed, and several modifications that can be made to several molecules containing the MSC secretome are discussed, specifically intended are all possible combinations and permutations of the MSC secretome, and modifications that are possible unless specifically indicated in the opposite sense. Therefore, if molecular classes A, B, and C, as well as molecular classes D, E, and F, and example molecular combination AD are disclosed, even if each is not individually enumerated, it is construed individually and collectively that combinations AE, AF, BD, BE, BF, CD, CE, and CF are disclosed. Similarly, any subset or combination thereof is also disclosed. For example, the subgroups AE, BF, and CE are considered to be disclosed. This concept applies to all aspects of this application, including but not limited to steps in a method for preparing and using the compositions of this disclosure. Therefore, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed by any particular embodiment or combination of embodiments of the method of this disclosure.

[0047] The primary nutritional characteristics of MSCs are the secretion of growth factors and exosomes that induce cell proliferation and angiogenesis. Exosomes express mitotic proteins such as transforming growth factor-alpha (TGF-α), TGF-β, hepatocyte growth factor (HGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), and insulin-1-like growth factor-1 (IGF-1). These promote the division of fibroblasts, epithelial cells, and endothelial cells. Vascular endothelial growth factor (VEGF), IGF-1, EGF, and angiopoietin-1 are released to recruit endothelial cells and initiate angiogenesis. MSCs support this process through paracrine mechanisms and regulate the regenerative environment through anti-inflammatory and immunomodulatory mechanisms. In response to inflammatory molecules such as interleukin-1 (IL-1), IL-6, IL-2, IL-12, tumor necrosis factor-α (TNF-α), and interferon-gamma (INF-γ), MSCs secrete an array of growth factors and anti-inflammatory proteins with complex feedback mechanisms among many types of immune cells. Key immunomodulatory cytokines include prostaglandin 2, TGF-131, HGF, SDF-1, nitrous oxide, indoleaniline 2,3-dioxygenase, IL-4, IL-10, IL-1 receptor antagonists, and soluble tumor necrosis factor-α receptors. MSCs interfere with the proliferation and function of many inflammatory immune cells, including T cells, natural killer cells, B cells, monocytes, macrophages, and dendritic cells. While MSCs across species can modulate T cell activity, these mechanisms are not identical across mammalian species.

[0048] A characteristic feature of a chronic inflammatory environment is a persistent imbalance in helper T cell and macrophage types. MSC exosomes indirectly promote the transition from TH1 to TH2 cells by reducing INF-γ and increasing IL-4 and IL-10. Restored TH1 / TH2 balance has been shown to improve tissue regeneration in cartilage, muscle, and other soft tissue injuries, alleviate symptoms of autoimmune diseases, and exert anti-diabetic effects. Similarly, reduced INF-γ and increased IL-4 secretion promote the shift of macrophages from M1 (pro-inflammatory, anti-angiogenic, and tissue growth inhibitory) to M2 (anti-inflammatory, pro-remodeling, and tissue healing) types, which are necessary for the healing and regeneration of the skeleton, muscle, and nerves.

[0049] This specification discloses complex compositions of extracellular vesicles containing secreted biomolecules (proteins, lipids, and ribonucleic acids) and / or biomolecules derived from mesenchymal cells. In one embodiment of this specification, a composition is disclosed comprising a therapeutically effective amount of MSC secretome (e.g., a composition comprising MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles) and one or more biomolecules (e.g., peptides, polypeptides, proteins, siRNAs, shRNAs, and / or microRNAs (miRNAs)).

[0050] In some embodiments, the therapeutic composition includes the following proteins: ferritin, NUP85, LAMP2, GPR115, serpine Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF R-alpha, soltirin, serpine B6, Dkk-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20 R-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecane-4 (one or more), as well as: Ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-IRA, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirre13, FLRT1, Galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurograinin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLIN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 Alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Me r, TREM-2, Flt-3L, CDS, IL-6, CD229, insulin, syntaxin 6, GRO, Bcl-w, lipocalin-2, PDGF-AA, IL-2 Ra, angiogenin, LYVE-1, CD4, RAGE, CDNF, brevican, NAP-2, PU.1, EDAR, ADAMTS13, kynureninase, PTH1R, IFN-gamma-R1, CrkL, B7-1, PARC, draxin, VE-cadherin, procalcitonin, SOX15, kallikrein-11, BCMA, dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, syndecane-4, IL-2 Rg, MICA, Dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, Glycoprotein V, Semaphorin 4G, IL-12p40, PSA-Total, IL-15, MAP1D, Clq, TNF4, Dtk, Endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, Galectin-8, CA4, Cystatin EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S10 Any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more of these proteins, or all of these proteins.

[0051] Extracellular vesicles (EVs) are small, membrane-bound spheres containing proteins and RNA (of which exosomes are a subset). Exosomes are small-diameter (e.g., 20–150 nm) lipid bilayer vesicles secreted by cells to enable paracrine signaling. Other EV populations originate directly from the plasma membrane or are formed during apoptosis (apoptotic bodies). This specification discloses compositions containing therapeutically effective amounts of MSC secretomes (e.g., compositions comprising MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles). In some embodiments, the therapeutic composition includes the following nucleic acids: HSA-LET-7A-5P, HSA-LET-7B-5P, HSA-LET-7C-5P, HSA-LET-7D-3P, HSA-LET-7E-5P, HSA-LET-7G-5P, HSA-LET-7I, HSA-LET-7I-5P, HSA-MIR-100-5P, HSA-MIR-103a-3P, HSA-MIR-106a-5 p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hs a-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, h sa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-m iR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-m ir-16-2, hsa-miR-16-5p, hsa-miR-1'7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-m iR-193b-3p, hsa-miR-19'7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p,hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-24-3p hsa-mir-25 hsa-miR-25-3p hsa-miR-26a-5p hsa-miR-27a-3p hsa-mir-27b hsa-miR-27b-3p hsa-miR-29a-3p hsa-miR-29c-3p hsa-miR-30a- 5p、hsa-miR-30a-5p、hsa-miR-30b-5p、hsa-miR-30c-5p、hsa-mir-30d、hs a-miR-30d-5p、hsa-mir-30e、hsa-miR-30e-5p、hsa-miR-31-3p、hsa-miR-3 1-5p, hsa-miR-320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3p, hsa-miR-376c-3p, hsa-miR-423-3p hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p, hsa-mir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a- 5p、およびhsa-miR-99b-5pのうち1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、 17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、40、45、50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more in any combination, or containing all of these nucleic acids.

[0052] Exemplary microRNA contents may include human miRNA sequences hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-214-3p, and hsa-miR-27a-3p, all of which have binding sites in TMPRSS2 mRNA.

[0053] In some embodiments, the therapeutic composition is CD63 + CD9 - CD81 - The composition contains extracellular vesicles having the phenotype of CD63. In some embodiments, at least 70, 75, 80, 85, 90, 91, 92, 93, 94, or 95% of the extracellular vesicles in the therapeutic composition are CD63. + CD9 - CD81 - In some embodiments, at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, or 95% of the extracellular vesicles in the therapeutic composition are CD9 - In some embodiments, at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, or 95% of the extracellular vesicles in the therapeutic composition are CD81. - That is the case.

[0054] In some embodiments, MSCs cultured to produce therapeutic compositions have the ability to undergo tricellular differentiation in vitro toward adipocyte, osteoblast, and chondrocyte phenotypes. In some embodiments, MSCs are positive for CD73, CD105, CD166, and CD90, and negative for CD14, CD31, CD34, and CD45.

[0055] The MSC secretome is understood and intended herein to include exosomes and growth factors. The growth factors and exosomes may be allogeneic or autologous. The growth factors and exosomes may originate from any cell in the human body, such as ectoderm cells, endoderm cells, or mesoderm cells. For example, the MSC secretome may include mesenchymal stem cell (MSC)-derived growth factors, MSC-derived exosomes, or both MSC-derived growth factors and exosomes. In some embodiments, the method further includes the step of adding at least one additive together with the exosomes and growth factors. Specifically, MSCs under appropriate wound healing conditions may produce suitable therapeutic agents, such as exosomes and growth factors, that can provide treatment for inflammatory lung diseases. In one embodiment herein, the MSC secretome composition comprises prostaglandin E2 (PGE2), transforming growth factor 131 (TGF-I31), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitrate oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TMF-α receptor, insulin-like growth factor, fibroblast growth factor (FGF) 1-23 (Patent A composition is disclosed further comprising FGF1 and FGF2), bone morphogenetic proteins (BMPs) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) macrophage-stimulating protein (MSP), platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF estrogen), and / or thyroid hormones.

[0056] Embodiments of therapeutic compositions described herein may include proteins and microRNAs, some of which may be embedded in or surrounded by lipid membranes to form vesicles ranging in size from approximately 20 nm to approximately 200 nm. The number of vesicles in the composition may be between approximately 1 million and 100 billion per mL when suspended, or between approximately 10 million and 1 trillion when formulated as a lyophilized powder.

[0057] A. Delivery of pharmaceutical carriers / medicines The therapeutic compositions described herein may be administered in vivo in a pharmaceutically acceptable carrier. “pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable; that is, the material can be administered to a subject together with a nucleic acid or vector without causing any undesirable biological effects or adverse interactions with any other components of the pharmaceutical composition containing the material. The carrier is, of course, selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject, as is well known to those skilled in the art. The composition may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, or topically, including by intranasal administration or inhalation. As used herein, “intranasal administration” means delivery of the composition to the nose and / or nasal passages through one or both nostrils, and may include delivery by spray or droplet mechanisms, or by aerosolization of the nucleic acid or vector.

[0058] The composition can be administered by inhalation via the nose or mouth, for example, by delivery using a spray or droplet mechanism such as a metered-dose inhaler, dry powder inhaler, nebulizer, or vaporization device. Delivery can also be directly to any area of ​​the respiratory system (e.g., the lungs) via intubation. The exact amount of composition required varies from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the disorder being treated, and the mode of administration.

[0059] Parenteral administration of a composition, when used, is characterized by injection as a whole. Injectable preparations can be prepared conventionally as a liquid solution or suspension, as a solid suitable for solution of a suspension in a liquid before injection, or as an emulsion. Parenteral administration may involve the use of a sustained-release or continuous-release system such that a constant dose is maintained.

[0060] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th edition) ed. ARGennaro, Mack Publishing Company, Easton, PA 1995. Typically, the formulation is made isotonic by using an appropriate amount of a pharmaceutically acceptable salt in the formulation. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Furthermore, the carrier comprises a sustained-release preparation such as a semipermeable matrix of a solid hydrophobic polymer containing the antibody, the matrix being in the form of a molded article, e.g., a film, liposomes, or microparticles. It will be obvious to those skilled in the art that the specific carrier may more preferably depend, for example, on the route of administration and the concentration of the composition being administered.

[0061] Pharmaceutical carriers are known to those skilled in the art. These are, most typically, standard carriers for drug administration to humans, including solutions such as sterile water, physiological saline, and buffered solutions of physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0062] The pharmaceutical composition may contain, in addition to the selected molecule, a carrier, a thickener, a diluent, a buffer, a preservative, a surfactant, and the like. The pharmaceutical composition may also contain one or more active ingredients such as an antibacterial agent, an anti-inflammatory agent, or an anesthetic.

[0063] The pharmaceutical composition may be administered in several ways, depending on whether topical or systemic treatment is desired and the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, and intranasal administration), oral, inhalation, or parenteral administration, such as intravenous infusion, subcutaneous injection, intraperitoneal injection, or intramuscular injection. The antibodies of this disclosure may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarially, or transdermally.

[0064] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions, or suspensions, including saline and buffering media. Parenteral vehicles include sodium chloride solutions, ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's solution, or fixative oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), etc. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases. Preparations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders. Conventional pharmaceutical carriers, aqueous bases, powder bases, or oily bases, thickeners, etc., may be essential or desirable.

[0065] Compositions for oral administration may include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing agents, or binders may also be desirable.

[0066] Possibly, portions of this composition may be administered as pharmaceutically acceptable acid addition salts or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, as well as organic bases such as mono-, di-, trialkyl and arylamines and substituted ethanolamines.

[0067] B. Therapeutic use Effective dosages and schedules for administering this composition can be determined empirically, and making such determinations is within the scope of the art. The dosage range for administering this composition should be large enough to produce the desired effect in which the symptoms of the disorder are affected. The dosage should not be large enough to cause adverse side effects such as unwanted cross-reactions or anaphylactic reactions. In general, the dosage can be determined by those skilled in the art, depending on the patient's age, condition, sex, and the severity of the disease, the route of administration, or whether other drugs are included in the regimen. The dosage may be adjusted by the individual physician if any contraindications exist. The dosage may vary and may be administered once or multiple times daily for one or several days, and / or once or multiple times monthly for two months or more, or once every one, two, or three months or more. Guidelines can be found in the literature on appropriate dosages for a given class of medicinal products.

[0068] C. Mesenchymal stem cells and therapeutic secretome compositions The therapeutic compositions disclosed herein utilize mesenchymal stem cell (MSC) secretomes and / or growth factors. In one embodiment herein, MSC secretome compositions (including, but not limited to, compositions comprising MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles) are disclosed. These therapeutic compositions may be used, for example, to treat, inhibit, reduce, alleviate, improve, and / or prevent conditions such as amyotrophic lateral sclerosis (ALS).

[0069] MSCs are pluripotent cells that have the ability to differentiate into a number of cell types, including muscle cells, chondrocytes, adipocytes, and osteoblasts. Typically, these cells can be found in the amniotic fluid, including the placenta, umbilical cord blood, adipose tissue, bone marrow, or perivascular tissue. As used herein, “MSC” refers to non-terminal differentiated cells, including but not limited to pluripotent stem cells, pluripotent stromal cells, stromal angiocytes, pericytes, perivascular cells, stromal cells, pluripotent cells, multipotent cells, adipocyte-derived fibroblast-like cells, adipocyte-derived stromal vascular fraction, adipocyte-derived MSCs, bone marrow-derived fibroblast-like cells, bone marrow-derived stromal vascular fraction, bone marrow-derived MSCs, tissue-derived fibroblast-like cells, adult stem cells, adult stromal cells, keratinocytes, and / or melanocytes.

[0070] In addition to their differentiation potential, MSCs possess immunomodulatory capabilities that result in the expression of many different cytokines and growth factors. As used herein, “MSC preparation” or “MSC secretome composition” refers to a composition comprising cell-free extracts of MSCs and / or MSC lysates obtained from human MSCs, fibroblast-like cells, and non-human animal MSCs, including but not limited to MSCs derived from horses, cattle, pigs, sheep, non-human primates, dogs, cats, rabbits, rats, and mice. In some embodiments, the MSCs may be derived from the patient to whom the composition is applied (autologous) or from another individual (allogeneic). MSCs may be collected in conditioned medium, cultured to increase the amount of cells relative to the lysate, or freshly used before being incorporated into the compositions of this disclosure. An MSC secretome composition (including, but not limited to, a composition comprising MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles) may contain about 0.00001 to about 20% by weight, for example, about 0.01 to about 10% by weight, of mesenchymal stem cell (MSC) extracts, MSC exosomes, or MSC growth factor preparations. The MSC preparation may contain either MSC-conditioned medium or MSC lysates from cell-cultured and grown MSCs. In some embodiments, the composition may further contain about 0.01 to about 10% by weight of cell-free medium conditioned by the growth of MSCs or MSC-lineage cells, where the cells are cultured under normal hyper-oxygenated culture conditions or artificial wound healing conditions.

[0071] As disclosed herein, MSCs used to produce the MSC additives of this disclosure (including growth factor secretome compositions which are either freeze-additives or powder-additives) can be selectively stimulated to produce MSC growth factors, secretomes, cytokines, chemokines, mesenchymal stem cell proteins, peptides, glycosaminoglycans, extracellular matrix (ECM), proteoglycans, secretomes, and exosomes. The growth factors and exosomes may be derived from any cell in the human body, such as ectoderm cells, endoderm cells, or mesoderm cells. As used herein, MSC growth factors include prostaglandin E2 (PGE2), transforming growth factor 131 (TGF-(31)), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitrate oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TMF-α receptor, insulin-like growth factor, fibroblast growth factor (FGF) 1-23 (in particular, FGF Examples of hormones include, but are not limited to, estrogen and thyroid hormones, as well as 1 and FGF2), bone morphogenetic proteins (BMPs) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), macrophage-stimulating protein (MSP), platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other hormones.

[0072] MSC culture may be carried out under wound healing and / or hypoxic conditions. Hypoxic conditions may include approximately 1% to approximately 5% oxygen, small or zero amounts of serum, small amounts of glucose, or various combinations of these elements. The combination of small amounts of nutrients and metabolites in the environment may induce tissue healing by inducing cultured cells to produce wound healing and anti-inflammatory ECM proteins and growth factors. Direct tissue healing is likely to be in the form of growth factors and cytokines, as well as novel ECM proteins such as collagen and glycosaminoglycans (GAGs). In one embodiment, an MSC preparation (e.g., an MSC secretome composition) includes MSC growth factors, MSC exosomes, and / or cell extracts of MSCs or MSC lysates obtained from MSCs cultured under standard hyper-oxygen culture conditions (e.g., 21% oxygen) or MSCs cultured under artificial wound healing conditions (e.g., 0.1% to approximately 5% oxygen).

[0073] As disclosed herein, artificial wound healing conditions simulate growth conditions in a real wound, where there is reduced nutrient supply and reduced waste removal that normally occurs due to disruption of local blood circulation. This creates a harsh environment for cells until new blood vessels are formed and blood circulation is restored. Therefore, artificial wound healing conditions used for culturing MSCs may include one or more of the following growth conditions: reduced glucose availability, reduced oxygen tension, reduced pH, and increased temperature.

[0074] In some embodiments, glucose availability can be reduced compared to a normal control (e.g., 4.5 g / L). In modified culture media that reduce glucose without damaging cells, glucose may be reduced by 0-50%, more preferably by about 5-40%. For example, under MSC artificial wound healing culture conditions, glucose may be reduced by about 5-15%, 10-20%, 15-25%, 20-30%, or 25-35%. In some embodiments, glucose may be present in a range of about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 g / L, or between any two of these values. In some embodiments, glucose is present at concentrations of less than or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5 g / L.

[0075] In some embodiments, the oxygen tension can be reduced to oxygen levels corresponding to hypoxic conditions. Normal atmospheric oxygen is approximately 21%, and levels below this are considered hypoxic. Therefore, in one embodiment, MSCs can be cultured at oxygen levels between 0.0% and 20.9%, between approximately 1% and 0.5%, between approximately 0.1% and 2.0%, between approximately 0.1% and 5.0%, between approximately 0.5% and 5.0%, between approximately 1.0% and 1%, between approximately 5.0% and 10.0%, and between approximately 10.0% and 15.0%. Hypoxic conditions can be a form of artificial wound healing conditions.Oxygen tension is between approximately 0.5% and 20.5% of oxygen when culturing MSCs to produce therapeutic secretome compositions containing extracellular vesicles and / or MSC secreted growth factors, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10 0.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15 This could be 0.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0% oxygen, or oxygen in the range between any two of these values.

[0076] The pH can also be lowered during MSC culture. The pH can be approximately 6.0 to 7.4, for example, 6.0 to 6.4, 6.2 to 6.4, 6.2 to 6.6, 6.4 to 6.6, 6.4 to 6.8, or 6.6 to 7.0, for example, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, or 7.4.

[0077] The temperature of the culture environment may be raised above the physiological homeostasis temperature (e.g., 37°C). In one embodiment, the culture conditions for MSCs may include approximately 35°C to 39°C, approximately 35°C to 36°C, approximately 36°C to 37°C, approximately 37°C to 38°C, approximately 38°C to 39°C, and approximately 39°C to 40°C. In one embodiment, the culture temperature may be 35.0, 35.1, 35.2, 35.3, 36.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36.0, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37.0, 37.1, 37.2, 37.3, 37.4, 37 The temperature can be 0.5, 37.6, 37.7, 37.8, 37.9, 38.0, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39.0, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, or 40.0℃.

[0078] In some embodiments, the culture medium is serum-free. In some embodiments, the serum-free culture medium contains platelet lysate. In some embodiments, the platelet lysate is human platelet lysate (HPL). In some embodiments, the serum-free culture medium contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% HPL by volume, or HPL in a range between any two of these values. In some embodiments, the culture medium contains 8%–12%, 5%–15%, or 9%–11% HPL by volume.

[0079] In one embodiment, an MSC secretome composition (including, but not limited to, a composition comprising MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles) may further include a protective coating (e.g., a cryoprotectant oligosaccharide and protein solution) that reduces the degradation of the growth factors. It is understood and intended herein that the protective coating may be designed as a polymer. "Polymer" refers to a relatively high molecular weight organic compound (natural or synthetic) whose structure can be represented by monomers, which are repeating small units. Non-limiting examples of polymers include polyethylene, rubber, and cellulose. Synthetic polymers are typically formed by addition polymerization or condensation polymerization of monomers. The term "copolymer" refers to a polymer formed from two or more different repeating units (monomer residues). By example, but not limited to, copolymers may be alternating copolymers, random copolymers, block copolymers, or graft copolymers. In certain embodiments, it is also intended that various block segments of a block copolymer themselves may constitute the copolymer. The term "polymer" encompasses all polymer forms, including but not limited to natural polymers, synthetic polymers, homopolymers, heteropolymers or copolymers, and addition polymers. In one embodiment, the gel matrix may include copolymers, block copolymers, diblock copolymers, and / or triblock copolymers. In one embodiment, the protective coating may include biocompatible polymers. In one embodiment, the biocompatible polymers may be crosslinked. Such polymers may also have the function of slowly releasing fat browning agents and / or fat modifiers into the tissue.As used herein, biocompatible polymers include polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyols); poly(olefin alcohols); polyvinylpyrrolidone); poly(hydroxyalkyl methacrylamide); poly(hydroxyalkyl methacrylate); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohols), polyhydroxy acids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid); poly-3-hydroxybutyrate or poly-4-hydroxy Examples include, but are not limited to, polyhydroxyalkanoates such as butyrates; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanone); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyether esters; polyacetals; polycyanoacrylates; polyacrylates; polymethyl methacrylates; polysiloxanes; poly(oxyethylene) / poly(oxyethylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid), and their copolymers.Biocompatible polymers also include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohol (PVA), methacrylate PVA (m-PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidones, polyglycolides, polysiloxanes, polyurethanes and their copolymers, alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic acid esters and methacrylate esters, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethylcellulose This may also include polycellulose, cellulose triacetate, sodium cellulose sulfate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohol), poly(vinyl acetate), polyvinyl chloride polystyrene and polyvinylpyrrolidone, their derivatives, their linear and branched copolymers and block copolymers, and their formulations.Examples of biodegradable polymers include polyesters, poly(orthoesters, polyethyleneamines), poly(caprolactone), poly(hydroxybutyrate), poly(hydroxyvalerate), polyanhydrides, poly(acrylic acid), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphospliazenes, their derivatives, their linear and branched copolymers and block copolymers, and their formulations.

[0080] In some embodiments, the protective coating comprises carbohydrate polymers such as monosaccharides, non-reducing polysaccharides, or disaccharides, and carbohydrate constructs of any combination thereof. Examples of carbohydrates that can be used in the protective coating include glucose, aldoses (such as D-allose, D-altrose, and D-mannose), glucopyranose, pentahydroxyhexanal, aD-glucopyranosyl-D-glucose, aD-glucopyranosyl-dihydrate, polymers of PD-glycopyranosyl units, PD-fructofuranosyl aD-glucopyranosyl (anhydrous / dihydrate), f3-D-galactopyranosyl-D-glucose, and aD-glucopyranosyl-aD-glucopyranosyl (anhydrous / dihydrate). It contains galactose, pentoses (ribose, xylose, lyxose), dextrose, dodecacarbon monodeca hydrate, fructose, sucrose, lactose, maltose, trehalose, agarose, D-galactosyl-0-(1-4)-anhydro-L-galactosyl, cellulose, PD-glycopyranosyl unit polymers, and starch, as well as polyhydric alcohols, polyalcohols, algitol, erythritol, glycitol, glycerol, xylitol, and sorbitol.

[0081] In some embodiments, the protective coating contains biocompatible and / or biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid). These particles may contain one or more of the following polyesters: glycolic acid units referred to herein as "PGA," lactic acid units referred to herein collectively as "PLA," such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide 5, caprolactone units referred to herein collectively as "PCL," such as poly(e-caprolactone), and copolymers referred to herein collectively as "PLGA," which include lactic acid units and glycolic acid units, for example, various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by a lactic acid:glycolic acid ratio, and their derivatives. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, collectively referred to herein as “PEGylated polymers,” such as various forms of PLGA-PEG or PEA-PEG copolymers. In certain embodiments, the “PEGylated polymer” can be obtained by covalently bonding the PEG region to the polymer via a cleavable linker. In one embodiment, the polymer contains at least 60, 65, 70, 75, 80, 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent of acetal pendant groups.

[0082] The triblock copolymers disclosed herein include core polymers such as polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), polyvinylpyrrolidone-co-vinyl acetate, polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic-glycolic) acid, poly(lactic-co-glycolic) acid (PLGA), and cellulose derivatives such as hydroxymethylcellulose and hydroxypropylcellulose. Examples of diblock copolymers that can be used in protective coatings disclosed herein include polymers such as polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), poly(vinylpyrrolidone-co-vinyl acetate), polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid), and poly(lactic acid-co-glycolic acid) (PLGA).

[0083] In one embodiment, the protective coating (i.e., the encapsulated composition) may further comprise lecithin or hydrolyzed lecithin as a carrier or encapsulating material. As used herein, lecithin and / or hydrolyzed lecithin coatings include coatings comprising phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid. The source of lecithin may be a plant or animal source.

[0084] In one embodiment, the polymer, monosaccharide, disaccharide, or polysaccharide used to form the protective coating formed by introducing the MSC additive into the encapsulation solution can be at a concentration suitable for forming the protective coating. For example, the polymer, monosaccharide, disaccharide, or polysaccharide can be at any concentration between 0.01 mM and 10.0 M, for example, about 0.01 M to about 0.1 M, about 0.1 mM to about 1.0 M, or about 1.0 M to about 10.0 M.

[0085] In one embodiment, the MSC secretome compositions disclosed herein (including, but not limited to, compositions comprising MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles) may contain any known components commonly found in the pharmaceutical field, such as free radical countermeasures, bactericides, metal ion chelating agents, preservatives, basicizing or acidifying agents, fragrances, surfactants, fillers, natural products or extracts of natural products, such as aloe or green tea extracts, vitamins, or coloring materials. Other components that may be combined with the powder may include antioxidants, which may be selected from a variety of antioxidants. Suitable antioxidants include vitamins, such as vitamin C (L-ascorbate, ascorbate-2-magnesium phosphate, ascorbyl palmitate, tetrahexyldecyl ascorbate), vitamin E (tocotrienol), vitamin A (provitamin A carotenoids such as retinol, retinal, retinoic acid, and beta-carotene), N-acetylglucosamine, or other glucosamine derivatives. Other components may include at least one essential fatty acid, such as S2-3, S2-6, and S2-9 polyunsaturated fatty acids, such as linoleic acid (LA), gamma-linoleic acid (GLA), alpha-linoleic acid (ALA), dihomo-γ-linoleic acid (DGLA), and arachidonic acid (ARA). Fatty acids may be derived from various sources, including evening primrose oil, blackcurrant oil, borage oil, or GLA-modified safflower seed oil. Other components may include a platelet-rich fibrin matrix, at least one component that supports ECM production and hyaluronic acid production, such as N-acetylglucosamine or other glucosamine derivatives, ultra-low molecular weight (ULMW) hyaluronic acid, chondroitin sulfate, or keratin sulfate.

[0086] The production of an MSC secretome composition may include culturing MSCs collected from a donor to create a culture medium under culture conditions containing reduced oxygen and nutrients (in some embodiments), stimulating the cultured cells to selectively secrete desired anti-inflammatory proteins, peptides, glycosaminoglycans, proteoglycan exosomes, and secretomes by adjusting cell growth conditions, collecting the aggregate mixture and combining it with a mounting solution, freezing the aggregate mixture containing exosomes, peptides, proteins, cytokines, growth factors, extracellular matrix (ECM), proteoglycans, glycosaminoglycans, and chemokines selected from the group consisting of human MSCs, animal MSCs, pluripotent stromal cells, fibroblasts, and fibroblasts, freezing the aggregate mixture in combination with a mounting solution such as an oligosaccharide such as a trehalose solution or a protein solution, and freeze-drying the frozen mixture to produce a dry powder. Alternatively, MSCs may be lysed in such a way that all MSCs are collected from the culture process, an extract lysate is produced, the extract lysate is concentrated, and the extract lysate is combined with a mounting solution such as an oligosaccharide such as a trehalose solution or protein solution, the mixture is frozen, and the frozen mixture is freeze-dried or lyophilized to produce a dry powder. The powder contains a highly concentrated aggregate of analgesic MSC secretomes and exosomes, as well as extracellular matrix components specific to anti-inflammatory properties.

[0087] This method may also include filtering, sterilizing, concentrating, freezing, or lyophilizing the MSC-conditioned culture medium. Furthermore, the MSC culture medium may be combined with a cryoprotectant before freezing.

[0088] Various methods exist for lysing MSCs. Lysis can be achieved by adding a hypotonic solution or by repeated freeze-thaw processes that disrupt the cell membrane. Furthermore, cells may be lysed while attached to the culture surface or in suspension. Cells may also be enzymatically released and / or lysed by mechanical homogenization.

[0089] Stimulating MSCs to selectively secrete desired anti-inflammatory proteins, peptides, glycosaminoglycans, proteoglycans, exosomes, and secretomes can be achieved by regulating cell growth conditions such as cell density, culture medium supplements, nutrient supplements, oxygen levels, length of culture under conditions, cell passage number, and combinations thereof.

[0090] III. Treatment methods for amyotrophic lateral sclerosis (ALS) In some embodiments, the therapeutic compositions disclosed herein are used in methods for treating a target amyotrophic lateral sclerosis (ALS). Any of the therapeutic compositions described herein may be used in such methods. ALS is a complex, progressive, and fatal neurodegenerative disorder that affects motor neurons, which die (atrophy) over time, leading to muscle weakness, loss of muscle mass, and loss of motor control, and ultimately death due to respiratory failure.

[0091] In some embodiments, the subjects have spinal-onset ALS. In some embodiments, the subjects have medullary-onset ALS. The subjects may also have progressive ALS. The subjects may exhibit limb-related symptoms. The subjects may exhibit dysphagia or difficulty speaking.

[0092] In some embodiments, treatment with the therapeutic compositions disclosed herein slows the progression of ALS.

[0093] In some embodiments, gene mutations may be associated with ALS. For example, mutations in superoxide dismutase type 1 (SOD1) have been found in ALS patients. In another example, a six-nucleotide hexanucleotide repeat extension, i.e., GGGGCC (G4C2), in the C9ORF72 gene is considered to be associated with familial ALS. In some embodiments, subjects with ALS may also have mutations in the SOD1 gene or the C9ORF72 gene, which can result in amino acid mutations in the SOD1 protein or the C9ORF72 protein, respectively. In some embodiments, subjects with ALS have one or more amino acid mutations in the SOD1 protein. For example, subjects with ALS may have one or more amino acid mutations including K3E, A4V, W32*, G38R, G41S, G72S, N86S, D90A, G93A, S105L, D109Y, C111Y, I112M, L126*, N139D, L144S, or combinations thereof, where * indicates cleavage due to the introduction of an early stop codon in the SOD1 protein. In some embodiments, subjects with ALS may have G4C2 proliferation in the C9ORF72 gene, which produces a longer form of the C9ORF72 protein containing dipeptide repeats. Non-restrictive dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, and poly-PR. In some embodiments, mutations in the SOD1 or C9ORF72 gene may be located in non-coding regions (e.g., introns).

[0094] In some embodiments, the ALS Functional Assessment Scale-R (ALSFRS-R), a questionnaire-based scale, can be used to measure and track changes in the physical function of ALS patients over time. The ALSFRS-R measures 12 aspects of physical function, including speech, salivation, swallowing, writing, food cutting, climbing stairs, turning over in bed, walking, grooming and hygiene, shortness of breath (dyspnea), orthostatic breathing (shortness of breath while lying down), and respiratory failure. Each function is scored from 4 (normal) to 0 (incapable), with a maximum total score of 48 and a minimum total score of 0. For example, a patient with a high ALSFRS-R score across all 12 aspects is considered to have more physical function. In some embodiments, the ALSFRS-R can be administered by a healthcare provider.

[0095] In some embodiments, the subject's ALSFRS-R score may increase after treatment with the therapeutic composition described herein. For example, the subject's ALSFRS-R score may increase by about 0.1 points per month after treatment with the therapeutic composition described herein, compared to the ALSFRS-R score measured before treatment. In some embodiments, the subject's ALSFRS-R score may increase by at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration of the therapeutic composition described herein, compared to the ALSFRS-R score measured before administration. In some embodiments, the ALSFRS-R score of the subject may increase by at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration of the therapeutic composition described herein, compared to the ALSFRS-R score measured before administration, for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more than 12 months.

[0096] In some embodiments, the ALSFRS-R score of the subject may decrease after treatment with the therapeutic composition described herein. For example, the ALSFRS-R score of the subject may decrease by less than approximately 0.3 points per month after treatment with the therapeutic composition described herein compared to the ALSFRS-R score measured before treatment. In some embodiments, the ALSFRS-R score of the subject may decrease by less than approximately 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration of the therapeutic composition described herein compared to the ALSFRS-R score measured before administration. In some embodiments, the target ALSFRS-R score decreases by less than approximately 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration of the therapeutic composition described herein, compared to the ALSFRS-R score measured before administration, for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more than 12 months.

[0097] In some embodiments, subjects may have a history of a decrease in their ALSFRS-R score prior to administration of the therapeutic MSC secretome composition. For example, a subject may have a history of a monthly decrease in their ALSFRS-R score of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 points prior to administration of the therapeutic MSC secretome composition.

[0098] This specification also discloses a method for treating a target ALS, comprising the step of administering to the target a composition comprising secretory extracellular vesicles containing any combination of proteins and / or miRNAs selected from: ferritin, NUP85, LAMP2, GPR115, serpin Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF R-alpha, soltirin, serpin B6, Dkk-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20 One or more of the following: R-beta, SREC-II, JAM-C, TNF-RI, htPAPP-A, eNOS, MSP-R, TPP1, LAMP1, B2M, NCAM-1, HIF-1-alpha, ST6GAL1, CD99-L2, Plexin-A4, EMMPRIN, p53, Semaphorin-7A, NKp80, Cystatin-B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin-L, RBP4, Serpine-A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP-alpha, and Syndecane-4, as well as: Ferritin, IGFBP-4, IL-1-R6, GSTM1, NUP85, LAMP2, Meprin-A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-IRA, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFA5, Galectin-9, vWF-A2, TACE, Activin RUB, Cathepsin S, LDL R, BMPR-IA, OX40, IL-3 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDT5, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-bl, FABP4, RGM-A, RELT, TrkC, C5a, SREC-I, Nestin, TPO, ErbB3, Kirrel3, FLRT1, Galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurograinin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RH, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 MI, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp 46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bcl-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.1, EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-Gamma-R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein-11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 At least one protein selected from the group consisting of Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, or IL-13, hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-1 00-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10 b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a- 3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p,hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1'7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p hsa-miR-193b-3p, hsa-miR-19'7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa -miR-222-3p、hsa-miR-22-3p、hsa-miR-23a-3p、hsa-miR-23b-3p、hsa-mir-24-1、hsa-mir-24-2、hsa-miR-24-3p、hsa-mir-25、hsa-miR-25-3p、hsa-miR-26a-5p、hsa-miR-27a-3p、hsa-mir-27b、hsa-miR-27b-3p、hsa-miR-29a-3p、hsa-miR-29c-3p、hsa-miR-30a-5p、hsa-miR-30a-5p、hsa-miR-30b- 5p、hsa-miR-30c-5p、hsa-mir-30d、hsa-miR-30d-5p、hsa-mir-30e、hsa-m iR-30e-5p、hsa-miR-31-3p、hsa-miR-31-5p、hsa-miR-320a、hsa-miR-342- 3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5phsa-miR-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p, hsa-mir -92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, and hsa-miR-99b-5p. ,

[0099] In some embodiments, the therapeutic product is administered in a cellular equivalent dose range of 0.7 to 7 million cells per kg. In some embodiments, the therapeutic product is administered in a cellular equivalent dose of at least about, at most about, or about 200,000, 500,000, 700,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 10,500,000, 11,000,000, 11,500,000, or 12,000,000 cells per kg. In some embodiments, the product is administered in a cellular equivalent dose of 9 × 10⁶ 11 ~1.2 × 10 12 Individual extracellular vesicles, or 5 × 10 11 ~1.5×10 12 , 6×10 11 ~1.4×10 12 , 7×10 11 ~1.3 × 10 12 , 8×10 11 ~1.2 × 10 12 , or 8 x 10 11 ~1.3 × 10 12 It is administered in a dose that yields 5 × 10⁶ extracellular vesicles. In some embodiments, the product yields at least or at most 5 × 10⁶ of the product. 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1 x 10 12 , 1.1 × 10 12 , 1.2 × 10 12 , 1.3 × 10 12 , 1.4×1012 , or 1.5 × 10 12 It is administered in a dose that provides 6 × 10⁶ extracellular vesicles per ml. In some embodiments, the therapeutic product is 6 × 10⁶ per ml. 10 ~8×10 10 , 5×10 10 ~9×10 10 , 4×10 10 ~10×10 10 , 5.5×10 10 ~8.5×10 10 , or 6×10 10 ~8.5×10 10 Contains 10 cells. In some embodiments, the therapeutic product contains 6 × 10 cells per ml. 10 ~8×10 10 It contains 6 × 10⁶ extracellular vesicles or cells and is administered in doses of 10-20 ml. In some embodiments, the therapeutic product is 6 × 10⁶ per ml. 10 ~8×10 10 It contains an extracellular vesicle or cell and is administered in doses of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml, or in a range between any two of these values.

[0100] In some embodiments, subjects that can be treated by the therapeutic products and methods described herein may include subjects who have ALS or have been diagnosed with ALS. In some embodiments, subjects may include mammals. In some embodiments, subjects may include humans. In some embodiments, subjects may include non-human mammals. Non-limiting examples of non-human mammals include non-human primates such as chimpanzees, other apes and monkey species, domestic animals such as cattle, horses, sheep, goats, and pigs, household animals such as rabbits, dogs, and cats, and laboratory animals such as rats, mice, and guinea pigs. [Examples]

[0101] The following examples are provided to provide a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are prepared and evaluated, and are intended to be purely illustrative but not intended to limit the disclosure. While efforts have been made to ensure accuracy with respect to numerical values ​​(e.g., quantities, temperatures, etc.), some error and deviation should be considered. These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0102] A. Example 1 - Production of therapeutic composition The MSC secretome therapeutic composition was prepared by the following method: Human bone marrow-derived MSCs were cultured in a culture vessel containing growth medium to expand the MSC population. The growth medium was then removed and the cells were washed with PBS. Subsequently, the MSCs were cultured in reduced glucose medium with a pH of less than 7.0 under hypoxic conditions. The conditioned medium was then collected, ultrafiltered, and filler-sterilized. The production process of the therapeutic product was carried out in accordance with current Good Manufacturing Practices and current Good Tissue Practices.

[0103] The tetraspanin profile of extracellular vesicles present in the therapeutic composition was determined, and it was found that over 95% of the extracellular vesicles present in the therapeutic composition were CD63. + CD9 - CD81 - They discovered that...

[0104] The protein content of the therapeutic product was determined, and the following proteins were identified: ferritin, NUP85, LAMP2, GPR115, serpine Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF R-alpha, sorbitin, serpine B6, Dkk-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20 R-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecane-4 (one or more), as well as: Ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-IRA, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, Nestin, TPO, ErbB3, Kirre13, FLRT1, Galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurograinin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLIN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 Alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, insulin, syntaxin 6, GRO, Bcl-w, lipocalin-2, PDGF-AA, IL-2 Ra, angiogenin, LYVE-1, CD4, RAGE, CDNF, brevican, NAP-2, PU.1, EDAR, ADAMTS13, kynureninase, PTH1R, IFN-gamma-R1, CrkL, B7-1, PARC, draxin, VE-cadherin, procalcitonin, SOX15, kallikrein-11, BCMA, dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, syndecane-4, IL-2 We found the presence of at least one protein selected from Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, and IL-13.

[0105] Determine the nucleic acid content of the therapeutic product, and the following nucleic acids: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1’7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-19’7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p,hsa-miR-26a-5p, hsa-miR-27a-3p, hsa-mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR -29c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-mir-30d , hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR- 320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3 p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa- mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-66 We found that the following compounds exist: 3a, hsa-miR-874-3p, hsa-mir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, and hsa-miR-99b-5p.

[0106] Example 2 - Pilot safety study of the treatment of amyotrophic lateral sclerosis using an investigational drug derived from extracellular vesicles of human bone marrow stem cells. In this example, the inventors have reported that an investigational drug (IP) of extracellular vesicles derived from human bone marrow stem cells (hBM-MSC EVs) is safe and effective in patients with amyotrophic lateral sclerosis (ALS). Ten ALS patients received two 10 mL intravenous (IV) infusions of the IP, one month apart, and were evaluated over three months. HBM-MSC EVs were considered safe for ALS patients. This initial trial suggests that controlled EV trials for the treatment of ALS are justified.

[0107] Amyotrophic lateral sclerosis (ALS) is a neurological disorder that affects the brain and spinal cord, causing loss of muscle control. Currently, there is no cure for ALS, and the disease worsens over time. Novel treatments using extracellular vesicles (MSC EVs) of mesenchymal stem cells are being tested. MSC EVs are small structures containing useful molecules and proteins that can be transported to disease-affected cells, helping to reduce inflammation and aid in repair. In this three-month trial, we examined the safety of human bone marrow MSC EVs (hBM-MSC EVs) administered as a treatment to 10 ALS patients, as well as how it acted to slow disease progression. We found that no serious side effects occurred with this treatment and that hBM-MSC EVs may have the potential to slow the progression of ALS. This indicates whether larger trials are needed to find details about the treatment, such as dosage (how much treatment is administered) and frequency (how often the treatment is administered), and how the treatment may relate to patient outcomes.

[0108] ALS is the third most common neurodegenerative disease after Alzheimer's disease and Parkinson's disease, and is the most common type of motor neuron disease. Despite an incidence of approximately 0.005%, there has been little progress in understanding and treating ALS. These diseases present in both spinal and bulbar forms. While genetically related causes have been defined, these events account for only a small number of cases. Therefore, it is hypothesized that less clearly defined environmental factors underlie 90%–95% of ALS cases, and until further research clarifies the causes, the latter cases are considered to have sporadic origins. The remaining 5–10% of cases with a clear genetic link to a family history are classified as familial, but no effective treatments have been developed to target this defined group. Patients are diagnosed based on symptom assessment through an exclusion process. Current research focuses on quantifying the role of protein waste products, namely TDP-43, SOD1, and FUS regulation and / or loss of clearance, the role of C9orf72 hexanucleotide repeat elongation, the most commonly associated mutations, and the contributions of other genes to the disease.

[0109] Riluzole, which lowers glutamate levels in neurons, is defined as an oral glutamatergic neurotransmitter inhibitor. Clinical trials have demonstrated limited results, extending patient survival by only 5-6 months. Edaravone, a potent antioxidant administered intravenously (IV), slows the progression of early-stage ALS, but recent publications have raised doubts about its findings in early clinical trials. Currently, these two are the only drugs approved by the FDA for the treatment of ALS, and access to these therapies may be limited and restricted. Given these limitations, the discovery and development of further treatments to halt or reverse the progression of both sporadic and familial ALS is urgently needed.

[0110] ExoFlo®, the investigational drug (IP) used herein, is an EV (hBM-MSC EV) preparation derived from human BM-MSCs. This IP is subject to advanced particle analysis, proteomics evaluation, and USP <71> It is a consistent extravasation product with extensive characterization, including sterility assurance. Furthermore, this IP is a biopharmacological product with consistent quality in terms of dose and biological activity through manufacturing in a cGMP environment. Two peer review studies have demonstrated the safety of intravenous administration of the IP to patients with severe COVID-19. The IP has also demonstrated efficacy in a subpopulation of these severely ill patients. The inventors hypothesized that the IP is safe for intravenous (IV) administration to patients with ALS and could potentially demonstrate efficacy in this patient population.

[0111] method Approval and Informed Consent. This study protocol was reviewed and approved by both the Institutional Review Board (IRS-001) and the Institutional Research Center for Cellular and Regenerative Medicine (IRCM) (JCRM-2021~296). In accordance with these approvals, an open-label, two-site, single-investigator pilot safety study was conducted on 10 subjects.

[0112] Study design. Participants met the following original inclusion criteria: 30–65 years of age, diagnosed with ALS, and obtained written informed consent from the participant or their legal representative. An IRB-approved protocol deviation was created, extending the age limit to 72 years to improve participant recruitment to 10. Participants were classified into spinal-onset (first presenting with limb involvement) or bulbar-onset (first presenting with dysphagia or dysarthria). Informed consent was obtained from all participants. ALS type was determined based on the primary symptom presentation of the disease, with the rate of decline being determined as rapid or normal. Spinal-onset ALS declined at an average rate of 1 point per month, while bulbar-onset ALS could decline much faster. Symptomatic time was defined as the time elapsed since the first reporting of ALS symptoms, and was typically much later than the onset of all related symptoms prior to diagnosis.

[0113] Safety was defined as the absence of adverse or serious adverse events related to the investigational drug (IP). For subject #5, who started the study with a revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score of 1, subjective assessments were performed by the patient's caregiver. As the patient was unable to move or speak, the assessment included communication through eye contact, concentration, and blinking.

[0114] Based on (1) the Phase I START trial using IV administration of BM-MSCs for acute respiratory distress syndrome (showing safety at up to 5 million cells per kg and a maximum dose of 10 million cells per kg), (2) observation of EV secretion of approximately 2,000 cells per cell, (3) laboratory analysis showing 6 to 80 billion EVs per mL, and (4) safe administration of a 10 mL dose to one past ALS patient, the IP dose was calculated. This showed a maximum IV IP dose of 17.5 mL for a 70 kg adult, and 10 mL of IV IP was determined to be a reasonable dose providing 0.6 to 0.8 trillion EV particles per dose.

[0115] Each participant underwent a baseline physical examination and assessment using the Revised Amyotrophic Lateral Sclerosis Functional Assessment Scale (ALSFRS-R score). Previous scores, if available in the participant's medical records, were recorded but not included in the assessment. The ALSFRS-R score was also recorded at the initial visit (time 0) immediately before intravenous administration of 10 mL of IP, and at the second visit one month later with IP infusion (time 1). Two additional follow-up visits (either in-person or virtual) were conducted at one-month intervals (times 2 and 3), resulting in a total of four visits over three months (see chart). The ALSFRS-R score was recorded at each visit and when documenting any adverse events observed after IP administration. Respiratory studies were not performed in this study. All 10 patients fully met the study protocol and completed the study.

[0116] Statistics. Repeated measures one-way ANOVA with Greenhouse-Geyser correction, assuming a Gaussian distribution of residuals, was performed on the data using GraphPad Prism 9.5.1 software. Simple linear regression analysis and gradient calculation for ALSFRS-R scores were also performed using Prism.

[0117] result Seven men and three women consented to the procedure. Riluzole was not administered to the subjects during the study. Seven of the subjects had spinal ALS. Of these spinal ALS subjects, three experienced accelerated progression based on reported functionality, with their ALSFRS-R scores decreasing by at least 3 points per month in the months leading up to the study. Three subjects had bulbar palsy. The age range of the subjects was 39–72 years, with a mean of 53.7 years. The time range from ALS diagnosis to the start of the study was 2–54 months, with a mean of 20.9 months, and half of the subjects had a history of the disease. Seven of the subjects were Caucasian, one was African American, and two were Asian (Indian and Armenian).

[0118] The baseline ALSFRS-R score ranged from 1 to 41 at time 0, with an average of 26.7 across all 10 subjects (Table 1). The scores at time 0 and time 1 were obtained immediately before the start of the first and second treatments, respectively. Each progressive score was recorded at one-month intervals over a continuous scoring period of three months (from time 0 to time 3). One subject had an abnormally high previous ALSFRS-R score of 1, but was included in this study to contribute to safety assessment across all stages of disease progression.

[0119] One-way ANOVA analysis of ALSFRS-R scores in all 10 subjects at all four time points showed no significant difference in the group mean at any of the four time points (F(1.2.36, 11.12)=4.06, p=0.062). The 9 subjects with no outliers had a baseline ALSFRS-R score range of 18-41, with a mean of 29.6. On the final day of the study, the ALSFRS-R score range for all groups was 1-42, with a mean of 23.6, while the group excluding outliers had a range of 13-42, with a mean of 26.1. This represents an average decrease of 3.1 points for all 10 subjects over the 3-month study period, and an average decrease of 3.5 points for the 9 subjects with no outliers.

[0120] Figure 1A shows the difference in raw ALSFRS-R scores across all subjects at each measurement period. Figure 1B shows fitted linear regression analysis over time for each subject to indicate disease progression during the study. One subject (S3) demonstrated a steady improvement in ALSFRS-R score, while the remaining two (S4 and S10) did not show a significant decrease in ALSFRS-R score. The remaining subjects showed a decline in score. From baseline (time 0) to the end of the study (36 months, time 3), the mean (+ / -SD) deltaFRS calculated from Table 1 across all 10 subjects was -1.03+ / -1.44. The mean slope derived from the linear regression analysis was -0.98+ / -1.67.

[0121] [Table 1]

[0122] Consideration Patients with ALS have no effective treatment options. Novel therapies are needed to reduce mortality and preserve function. MSC-derived extracellular viable cells (EVs) have the potential to offer a novel therapy based on preclinical and clinical evidence regarding safety and efficacy. Safety is a critical aspect of all early-stage trials of new investigational drugs for new disease indications. In this study, 10 ALS patients were treated with hBM-MSC EV IP to evaluate safety risks and potential efficacy. No adverse or serious adverse events related to the investigational drug were observed. This safety profile was consistent with the excellent safety profile observed with intravenous administration of up to two doses (15 ml each) of IP in patients with severe COVID-19. This small, open-label pilot safety study suggests that intravenous delivery of bone marrow-derived MSC EVs is safe for patients with ALS.

[0123] Recent studies have identified multiple candidate molecular, biochemical, and cellular mechanisms within the CNS and periphery, mediated by EV cargo miRNAs and proteins, regarding the possible mechanisms of hBM-MSC EV's efficacy against ALS. This multi-entity cargo can favorably modulate responses crucial to synaptic plasticity, neurogenesis, axonal growth, glial cell function, cellular apoptosis, immunomodulation, and other nervous system functions. For example, several miRNA species contained in BM-MSC EV can modulate the inflammatory, anti-inflammatory, and neurotoxic activities of astrocytes and / or microglia isolated from SOD1G93A mice (a mouse model of ALS), as well as motor neurons differentiated from inducible neural progenitor cells of ALS patients with SOD1G93A or C9orf72 mutations. The net effect of BM-MSC EV in in vitro and in vivo models is the reduction of both neurotoxicity and neuroinflammation. Furthermore, the roles of MSC EVs in regulating autophagy, cytoplasmic shuttling, and lysosomal flux, as well as the impairment of pre-synaptic vesicle dynamics caused by common C9orf72 ALS mutations, suggest that specific BM-MSC EV-related miRNAs may directly improve synaptic function.

[0124] Furthermore, BM-MSC EV's ability to cross the blood-brain barrier provides an opportunity to treat this end-stage disease using a safer intravenous administration approach. This ability allows for more convenient and frequent medication, helping to continuously enhance all possible gains from the available treatment options.

Claims

1. A method for treating amyotrophic lateral sclerosis (ALS) in a subject requiring treatment for ALS, comprising the step of administering to the subject a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles, wherein at least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 ＋ CD9 － CD81 － The method.

2. The method according to claim 1, wherein the ALS Functional Rating Scale-Revised (ALSFRS-R) score of the subject increases by at least approximately 0.1 points per month after administration, or decreases by less than approximately 3.0 points per month, compared to the ALSSFRS-R score measured before administration.

3. The method according to claim 2, wherein the ALSFRS-R score of the subject increases by at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration, compared to the ALSFRS-R score measured before administration.

4. The method according to claim 2, wherein the ALSFRS-R score of the subject decreases by less than approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration.

5. The method according to any one of claims 2 to 4, wherein the subject has a history of a decrease of approximately 3.0 points in the ALSFRS-R score per month prior to administration of the therapeutic MSC secretome composition.

6. A method for treating amyotrophic lateral sclerosis (ALS) in a subject requiring treatment for ALS, comprising the step of administering to the subject a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles, wherein the subject's ALS Function Rating Scale-R score increases by at least about 0.1 points per month after administration, or decreases by less than about 3.0 points per month, compared to the ALS FRS-R score measured before administration.

7. The method according to claim 6, wherein the ALSFRS-R score of the subject increases by at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration, compared to the ALSFRS-R score measured before administration.

8. The method according to claim 6, wherein the ALSFRS-R score of the subject decreases by less than approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration.

9. The method according to any one of claims 6 to 8, wherein the subject has a history of a decrease of approximately 3.0 points in the ALSFRS-R score per month prior to administration of the therapeutic MSC secretome composition.

10. At least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 ＋ CD9 － CD81 － The method according to any one of claims 6 to 9.

11. The therapeutic MSC secretome composition contains the following proteins: ferritin, NUP85, LAMP2, GPR115, serpin Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF-α-alpha, soltirin, serpin B6, DKK-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20-β-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecan-4 (one or more of these), as well as: Ferritin, IGFBP-4, IL-1 R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, DKK-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18, BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R-Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAM, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1, R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2, Rb, GFR Alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-Selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-lra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP (TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, NES Chin, TPO, ErbB3, Kirre13, FLRT1, galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-Alpha 1, AMIGO 2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 Alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, M er, TRIME-2, Flt-3L, CDS, IL-6, CD229, insulin, syntaxin 6, GRO, Bcl-w, lipocalin-2, PDGF-AA, IL-2 Ra, angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.

1. EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-Gamma-R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein-11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 The method according to any one of claims 1 to 10, further comprising at least one protein selected from Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin E M, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, or IL-13.

12. The extracellular vesicles contain the following nucleic acids: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-mir-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1’7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-19’7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-hsa-miR-27a-3p, hsa-mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR -30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-mir-30d, hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-342- 3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa-mir-486-1, hsa-mir-4 86-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p, hs The method according to any one of claims 1 to 11, comprising one or more of a-mir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, or hsa-miR-99b-5p.

13. The composition is (a) Under the following conditions: (i) oxygen tension less than 5%, and (ii) Culture medium with a pH of less than 7 So, the process involves culturing bone marrow-derived MSCs to produce MSC-conditioned medium, (b) A step of collecting the MSC-acclimatized medium, (c) A step of formulating the MSC-conditioned medium to produce the therapeutic MSC secretome composition, wherein the therapeutic MSC secretome composition comprises proteins and extracellular vesicles produced by the bone marrow-derived MSCs in step (a) The method according to any one of claims 1 to 12, produced by...

14. The method according to claim 13, wherein the culture medium is serum-free.

15. The method according to claim 13 or 14, wherein the glucose concentration of the culture medium is less than 4.5 g / L.

16. The method according to any one of claims 1 to 15, wherein the subject is suffering from spinal-onset ALS.

17. The method according to any one of claims 1 to 15, wherein the subject is suffering from medullary-onset ALS.

18. The method according to any one of claims 1 to 17, wherein the subject is suffering from progressive ALS.

19. The method according to any one of claims 1 to 18, wherein the subject exhibits limb-related symptoms.

20. The method according to any one of claims 1 to 19, wherein the subject exhibits dysphagia or difficulty speaking.

21. The method according to any one of claims 1 to 20, wherein the progression of ALS is slowed by the treatment.

22. The method according to any one of claims 1 to 21, wherein the subject has one or more amino acid mutations in the SOD1 protein.

23. The method according to claim 22, wherein the one or more amino acid mutations include G93A.

24. The method according to any one of claims 1 to 21, wherein the subject has one or more dipeptide repeats in the C9ORF72 protein.

25. The method according to claim 24, wherein the one or more dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

26. The method according to any one of claims 1 to 25, wherein the subject is a human.

27. The method according to any one of claims 13 to 26, wherein the bone marrow-derived MSCs are derived from human bone marrow.

28. The method according to any one of claims 1 to 27, wherein the administration step includes intravenous administration.

29. The method according to any one of claims 1 to 28, wherein the dose of the therapeutic MSC secretome composition administered to the subject is a cellular equivalent dose of 0.7 to 7 million cells per kg.

30. The therapeutic MSC secretome composition contains 4 × 10 per ml. １０ ~10 x 10 １０ The method according to any one of claims 1 to 28, comprising a single cell.

31. The therapeutic MSC secretome composition contains 5×10 １１ to 1.5×10 １２ extracellular vesicles, and the method according to any one of claims 1 to 28.

32. The method according to any one of claims 1 to 31, wherein the composition is administered monthly for two months or more, or once every one month, two months, or three months or more.

33. A method for preparing a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition for treating ALS in patients requiring treatment for amyotrophic lateral sclerosis (ALS), (a) Under the following conditions: (i) oxygen tension less than 5%, and (ii) Culture medium with a pH of less than 7 So, the process involves culturing bone marrow-derived MSCs to produce MSC-conditioned medium, (b) A step of collecting the MSC-acclimatized medium, (c) A step of formulating the MSC-conditioned medium to produce the therapeutic MSC secretome composition, wherein the therapeutic MSC secretome composition comprises proteins and extracellular vesicles produced by the bone marrow-derived MSCs in step (a) A method that includes this.

34. The method according to claim 33, wherein the culture medium is serum-free.

35. The method according to claim 33 or 34, wherein the glucose concentration of the culture medium is less than 4.5 g / L.

36. At least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 ＋ CD9 － CD81 － The method according to any one of claims 33 to 35.

37. The method according to any one of claims 33 to 36, wherein the bone marrow-derived MSCs are derived from human bone marrow.

38. The therapeutic MSC secretome composition contains the following proteins: ferritin, NUP85, LAMP2, GPR115, serpin Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF-α-alpha, soltirin, serpin B6, DKK-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20-β-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecan-4 (one or more of these), as well as: Ferritin, IGFBP-4, IL-1 R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, DKK-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18, BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R-Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAM, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1, R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2, Rb, GFR Alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-Selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-lra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP (TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, NES Chin, TPO, ErbB3, Kirre13, FLRT1, galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-Alpha 1, AMIGO 2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 Alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, M er, TRIME-2, Flt-3L, CDS, IL-6, CD229, insulin, syntaxin 6, GRO, Bcl-w, lipocalin-2, PDGF-AA, IL-2 Ra, angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.

1. EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-Gamma-R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein-11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 The method according to any one of claims 33 to 37, comprising at least one protein selected from Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin E M, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, or IL-13.

39. The therapeutic MSC secretome composition comprises the following nucleic acids: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-mir-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1’7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-19’7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p,hsa-miR-26a-5p, hsa-miR-27a-3p, hsa-mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-2 9c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-mir-30d, hsa- miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR-320a, hsa -miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3p, hsa-miR-3 76c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa-mir-486-1, hsa- mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p The method according to any one of claims 33 to 38, comprising one or more of hsa-mir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, or hsa-miR-99b-5p.

40. Use of a composition produced by the method of any one of claims 33 to 39 in the treatment of amyotrophic lateral sclerosis (ALS) in subjects requiring treatment for ALS.

41. The use according to claim 40, wherein the subject is suffering from spinal-onset ALS.

42. The use according to claim 40, wherein the subject is suffering from medullary-onset ALS.

43. The use according to any one of claims 40 to 42, wherein the subject is suffering from progressive ALS.

44. The use according to any one of claims 40 to 43, wherein the subject exhibits limb-related symptoms.

45. The use according to any one of claims 40 to 44, wherein the subject exhibits dysphagia or difficulty speaking.

46. The use according to any one of claims 40 to 45, wherein the treatment slows the progression of ALS.

47. The use according to any one of claims 40 to 46, wherein the subject has one or more amino acid mutations in the SOD1 protein.

48. The use according to claim 47, wherein the one or more amino acid mutations include G93A.

49. The use according to any one of claims 40 to 46, wherein the subject has one or more dipeptide repeats in the C9ORF72 protein.

50. The use according to claim 49, wherein the one or more dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

51. The method according to any one of claims 40 to 50, wherein the subject is a human.

52. The use according to any one of claims 40 to 51, wherein the composition is administered intravenously to the subject.

53. The use according to claim 52, wherein the ALS Functional Assessment Scale-Revised (ALSFRS-R) score of the subject increases by at least about 0.1 points per month after administration, or decreases by less than about 3.0 points per month, compared to the ALSSFRS-R score measured before administration.

54. The use according to claim 53, wherein the ALSFRS-R score of the subject increases by at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration, compared to the ALSFRS-R score measured before administration.

55. The use according to claim 53, wherein the ALSFRS-R score of the subject decreases by less than approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration.

56. The use according to any one of claims 40 to 55, wherein the subject has a history of a decrease of approximately 3.0 points in the ALSFRS-R score per month prior to administration of the therapeutic MSC secretome composition.

57. The use according to any one of claims 52 to 56, wherein the dose of the therapeutic MSC secretome composition administered to the subject is a cellular equivalent dose of 0.7 to 7 million cells per kg.

58. The therapeutic MSC secretome composition contains 4 × 10 per ml. １０ ~10 x 10 １０ The use according to any one of claims 52 to 56, comprising a single cell.

59. The therapeutic MSC secretome composition is 5 × 10 １１ ~1.5 x 10 １２ The use according to any one of claims 52 to 56, comprising a number of extracellular vesicles.

60. The use according to any one of claims 52 to 58, wherein the composition is administered monthly for a period of two months or more, or once every one month, two months, or three months or more.

61. The use of a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles in the treatment of amyotrophic lateral sclerosis (ALS) in subjects requiring treatment for ALS, wherein at least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 ＋ CD9 － CD81 － It is used.

62. The use according to claim 61, wherein the composition is administered intravenously to the subject.

63. The use according to claim 62, wherein the ALS Functional Rating Scale-Revised (ALSFRS-R) score of the subject increases by at least approximately 0.1 points per month after administration, or decreases by less than approximately 3.0 points per month, compared to the ALSSFRS-R score measured before administration.

64. The use according to claim 63, wherein the ALSFRS-R score of the subject increases by at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration, compared to the ALSFRS-R score measured before administration.

65. The use according to claim 63, wherein the ALSFRS-R score of the subject decreases by less than approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration.

66. The use according to any one of claims 61 to 65, wherein the subject has a history of a decrease of approximately 3.0 points in the ALSFRS-R score per month prior to administration of the therapeutic MSC secretome composition.

67. Use of a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles in the treatment of amyotrophic lateral sclerosis (ALS) in subjects requiring treatment for ALS, wherein the subject's ALS Function Rating Scale-R score increases by at least approximately 0.1 points per month after administration, or decreases by less than approximately 3.0 points per month, compared to the ALS FRS-R score measured before administration.

68. The use according to claim 67, wherein the ALSFRS-R score of the subject increases by at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 points per month after administration, compared to the ALSFRS-R score measured before administration.

69. The use according to claim 67, wherein the ALSFRS-R score of the subject decreases by less than approximately 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 points per month after administration, compared to the ALSFRS-R score measured before administration.

70. The use according to any one of claims 67 to 69, wherein the subject has a history of a decrease of approximately 3.0 points in the ALSFRS-R score per month prior to administration of the therapeutic MSC secretome composition.

71. At least 80% of the extracellular vesicles in the therapeutic MSC secretome composition are CD63 ＋ CD9 － CD81 － The use according to any one of claims 67 to 70.

72. The therapeutic MSC secretome composition contains the following proteins: ferritin, NUP85, LAMP2, GPR115, serpin Fl, OPN, PAI-1, DAPP1, cathepsin B, semaphorin 6C, PDGF-α-alpha, soltirin, serpin B6, DKK-3, thrombomodulin, PF4, MIF, periostin, furin, TIMP-1, decorin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, lumican, TIMP-2, SLITRK5, FAP, artemin, DPPII, cIAP-1, pentraxin 3, visfatin, neprilysin, albumin, galectin-1, UNC5H3, IL-20-β-beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midoquin, Calreticulin, Osteoactivin, Regmine, TAZ, Cathepsin L, RBP4, Serpine A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin-3, SIRP alpha, and Syndecan-4 (one or more of these), as well as: Ferritin, IGFBP-4, IL-1 R6, GSTM1, NUP85, LAMP2, Meprin A, IL-1 F10, bIG-H3, GPR115, TGFbl, Ephrin-A4, CD109, Serpine Fl, IGFBP-6, HS3ST4, Aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R-Alpha, Sortiline, Serpine B6, DKK-3, CNTF, TSP-1, GM-CSFRa, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Fulin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin Alpha 1, EXTL3, IL-18, BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRLS, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Parcephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R-Beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, ClqTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matryptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAKR, Nidogen-1, CD2, Kallikrein-1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAM, OSM R Beta, Thrombospondin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF -1 Alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Nuturin, Clement-2, EMMPRIN, Activin RIB, Neuroligin 2, Epilegulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Paparin-2, Syndecane-3, Jagged1, AKR1C4, Olfactomedine-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphAl, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin Alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA 125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Regmine, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 Beta, TAFA1, Podocalyxin, RalA, CRELD2, GRAP2, SP-D, BID, GFR Alpha-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1, R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Newdecine, ILT4, uPAR, Axl, WIF-1, IL-7 R-Alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Kokurin, Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGFR, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRKS, PD-1, Serpine A4, ADAM23, NOV, Galectin-2, Neurexin 3 Beta, TLR3, Sirtuin 2, Numb, IL-28 R Alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2, Rb, GFR Alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-Selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-lra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpine A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galectin-9, vWF-A2, TACE, Activin RIM, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2, NUDTS, ROR2, EphB4, Glypican 1, LAP (TGFb1), Gash, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10Rb, XEDAR, IL-22, PILR-alpha, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, NES Chin, TPO, ErbB3, Kirre13, FLRT1, galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R-Alpha 1, AMIGO 2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan, Syntaxin 4, VAMP-1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23R, Galectin-7, GALNT3, GITR L, CD14, R-Spongin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-RII, PGRP-S, SDF-la, PDGF-AB, GPVI, CD40, SCF R, Thrombospongin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan, Granulisin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin-3, ARSB, LIF R Alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP Alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-lb, NKp46, MCP-3, IL-32 Alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, M er, TRIME-2, Flt-3L, CDS, IL-6, CD229, insulin, syntaxin 6, GRO, Bcl-w, lipocalin-2, PDGF-AA, IL-2 Ra, angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.

1. EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-Gamma-R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein-11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndecan-4, IL-2 The use according to any one of claims 61 to 71, comprising at least one protein selected from Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, Clq, TNF4, Dtk, endoglin, ENA-78, Reg3A, MIP-lb, FGF-17, IL-6R, IL-8, galectin-8, CA4, cystatin E M, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, or IL-13.

73. The extracellular vesicles contain the following nucleic acids: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-1’7-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-�p, hsa-miR-193b-3p, hsa-miR-19’7-3p, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p, hsa-miR-26a-5p,hsa-miR-27a-3p, hsa-mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR- 30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-mir-30d, hsa-miR-30d-5p, h sa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR-320a, hsa-miR-342-3 p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3p, hsa-miR-376c-3p, hs a-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa-mir-486-1, hsa-mir-486 -2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p, hsa-m The use according to any one of claims 61 to 72, further comprising one or more of ir-92a-1, hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p, or hsa-miR-99b-5p.

74. The composition is (a) Under the following conditions: (i) oxygen tension less than 5%, and (ii) Culture medium with a pH of less than 7 So, the process involves culturing bone marrow-derived MSCs to produce MSC-conditioned medium, (b) A step of collecting the MSC-acclimatized medium, (c) A step of formulating the MSC-conditioned medium to produce the therapeutic MSC secretome composition, wherein the therapeutic MSC secretome composition comprises proteins and extracellular vesicles produced by the bone marrow-derived MSCs in step (a) The use according to any one of claims 61 to 73, produced by...

75. The use according to claim 74, wherein the culture medium is serum-free.

76. The use according to claim 74 or 75, wherein the glucose concentration of the culture medium is less than 4.5 g / L.

77. The use according to any one of claims 61 to 76, wherein the subject is suffering from spinal-onset ALS.

78. The use according to any one of claims 61 to 76, wherein the subject is suffering from medullary-onset ALS.

79. The use according to any one of claims 61 to 78, wherein the subject is suffering from progressive ALS.

80. The use according to any one of claims 61 to 79, wherein the subject exhibits limb-related symptoms.

81. The use according to any one of claims 61 to 80, wherein the subject exhibits dysphagia or difficulty speaking.

82. The use according to any one of claims 61 to 81, wherein the treatment slows the progression of ALS.

83. The use according to any one of claims 61 to 82, wherein the subject has one or more amino acid mutations in the SOD1 protein.

84. The use according to claim 83, wherein the one or more amino acid mutations include G93A.

85. The use according to any one of claims 61 to 82, wherein the subject has one or more dipeptide repeats in the C9ORF72 protein.

86. The use according to claim 85, wherein the one or more dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

87. The method according to any one of claims 61 to 86, wherein the subject is a human.

88. The use according to any one of claims 74 to 87, wherein the bone marrow-derived MSCs are derived from human bone marrow.

89. The use according to any one of claims 67 to 88, wherein the composition is administered intravenously to the subject.

90. The use according to any one of claims 61 to 89, wherein the dose of the therapeutic MSC secretome composition administered to the subject is a cellular equivalent dose of 0.7 to 7 million cells per kg.

91. The therapeutic MSC secretome composition contains 4 × 10 per ml. １０ ~10 x 10 １０ The use according to any one of claims 61 to 89, comprising individual cells.

92. The therapeutic MSC secretome composition is 5 × 10 １１ ~1.5 x 10 １２ The use according to any one of claims 61 to 89, comprising an extracellular vesicle.

93. The use according to any one of claims 61 to 92, wherein the composition is administered to the subject monthly for a period of two months or more, or once every one month, two months, or three months or more.

Images