Amyotrophic lateral sclerosis treatment using extracellular vesicle compositions

Abstract

Disclosed herein are methods of treating amyotrophic lateral sclerosis in a subject by administering a therapeutic MSC secretory group product prepared by a method comprising culturing a bone marrow-derived MSC under conditions comprising an oxygen tension of less than 5% and a culture medium having a pH of less than 7.

Description

Cross-references

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 507,204, filed June 9, 2023, and U.S. Provisional Application No. 63 / 570,632, filed March 27, 2024, which are incorporated herein by reference in their entirety. Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a devastating terminal neurodegenerative disease affecting approximately 4.5 out of every 100,000 people. ALS is the most common type of motor neuron disease and the third most common neurodegenerative disease after Alzheimer's and Parkinson's. ALS is a neurological disorder affecting the brain and spinal cord, leading to loss of muscle control. While approximately 4.5 out of every 100,000 people live with the disease, unfortunately, progress in understanding and treating ALS is minimal. Current diagnosis is based on symptom-based exclusion. Currently, there is no cure for ALS, and the disease worsens over time. Unfortunately, there are no drug treatments to stop or reverse the progression of ALS, and access to these treatments can be limited and restricted. There is a need for safe and effective treatments for ALS. Summary of the Invention

[0003] In some respects, this document discloses a method for treating amyotrophic lateral sclerosis (ALS) in a desired subject, the method comprising 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 - .

[0004] In some implementations, the subjects showed an increase of at least about 0.1 points per month in their ALSFRS-R score after application, or a decrease of less than about 3.0 points per month, compared to their ALSFRS-R score measured before application. In some implementations, the subjects showed an increase of 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 in their ALSFRS-R score after application, compared to their ALSFRS-R score measured before application. In some embodiments, the subjects showed a monthly decrease in ALSFRS-R score of 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 after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subjects had a history of a monthly decrease in ALSFRS-R score of approximately 3.0 points prior to administration of the therapeutic MSC secretome composition.

[0005] In some respects, this document discloses a method for treating amyotrophic lateral sclerosis (ALS) in subjects in need, the method comprising administering to the subject a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles, wherein, after administration, the subject has an increase of at least about 0.1 points per month in the ALS Functional Rating Scale-Revised (ALSFRS-R) score or a decrease of less than about 3.0 points per month in the ALS Functional Rating Scale-Revised (ALSFRS-R) score measured before administration.

[0006] In some implementations, the subjects showed an increase in their ALSFRS-R score 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 application, compared to the ALSFRS-R score measured before application. In some embodiments, the subjects showed a monthly decrease in ALSFRS-R score of 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 after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subjects had a history of a monthly decrease in ALSFRS-R score of approximately 3.0 points 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 comprises one or more of the following proteins: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulator protein, PF4, MIF, periosteal protein, furin, TIMP-1, trabeculin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20 Rβ, SREC-II, JAM-C, TNF. RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephricin, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF R α, Selectin, Serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Coagulation regulator protein, Endoglycosides, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostrin, Flintase, TIMP-1, PAPP-A, Glycerin proteoglycan, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lutein proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinal protein, ENPP-2, CD97, CTACK, Integrin α1, EXTL3, IL-18BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte Glycol-B3, FABP6, CHI3L1, FCRLS, TFF3, Nephlebotomycin, DPPII, cIAP-1, PDGF Rb, Pentanecoprotein 3, Angiotensinogen, Follicle-stimulating hormone-inhibin factor, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactone, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral Lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLP R, LAMP1, TROY, VCAM-1, Salivary lectin-11, S100A1, PAR1, Thyroid peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM Rβ, thromboretin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1(CD56), Granulase B, Lipocontin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, conglomerate A4, brain signaling protein 4D, ROBO2, PDX-1, APRIL, neuronal rank protein, transmembrane protein-2 with ring structure, EMMPRIN, activating protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGFR1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, Pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesenchymal protein-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGFR3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MISRII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 Rα, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator protein RIIA, ANG-2, Cochlin, progerin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease protein C, cysteine ​​protease protein D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A (Nogo-A), HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CFXIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactochonin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galactochonin-9, vWF-A2, TACE, Activator protein 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, phosphatidylinositol polysaccharide 1, LAP(TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-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 α1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, PIGF-2, neurogranulin, HE4, IL-23 R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-α, PGRP-S, SDF-1α, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1RII, neurotrophin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granulin, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIFR α, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein 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 one or more of 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-3p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, h sa-miR-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-57 4-3p, 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 or hsa-miR-99b-5p. ,

[0009] In some embodiments, the composition is produced as follows: (a) culturing bone marrow-derived MSCs under the following conditions to produce MSC conditioned medium: (i) oxygen tension below 5%; and (ii) a medium with a pH below 7; (b) harvesting the MSC conditioned medium; and (c) preparing 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). In some embodiments, the medium is serum-free. In some embodiments, the medium has a glucose concentration of less than 4.5 g / L.

[0010] In some embodiments, the subject has spinal-onset ALS. In some embodiments, the subject has medullary-onset ALS. In some embodiments, the subject has advanced ALS. In some embodiments, the subject presents with limb-related symptoms. In some embodiments, the subject presents with dysphagia or speech difficulties. In some embodiments, the treatment delays the progression of ALS.

[0011] In some embodiments, the object carries one or more amino acid variations of the SOD1 protein. In some embodiments, the one or more amino acid variations include G93A. In some embodiments, the object carries one or more dipeptide repeats of the C9ORF72 protein. In some embodiments, the one or more dipeptide repeats include poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

[0012] In some implementations, the object is a human being. In some implementations, the bone marrow-derived MSCs are derived from human bone marrow.

[0013] In some embodiments, administration includes intravenous administration. In some embodiments, the dose of the therapeutic MSC secretome composition administered to the subject is a cell equivalent dose of 0.7 to 7 million cells / kg. In some embodiments, the therapeutic MSC secretome composition comprises 4 x 10 10 Up to 10x10 10 Cells / ml. In some embodiments, the therapeutic MSC secretome composition contains 5 x 10 cells / ml. 11 Up to 1.5x10 12 One extracellular vesicle. In some embodiments, the composition is applied monthly for two or more months, or once every 1, 2, or 3 months or more.

[0014] In some aspects, this document discloses a method for preparing a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition for treating amyotrophic lateral sclerosis (ALS) in subjects of need, the method comprising: (a) culturing bone marrow-derived MSCs to produce an MSC conditioned medium under the following conditions: (i) oxygen tension below 5%; and (ii) a medium with a pH below 7; (b) harvesting the MSC conditioned medium; and (c) 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 of step (a).

[0015] In some embodiments, the culture medium is serum-free. In some embodiments, the culture medium has a glucose concentration of 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 implementations, the bone marrow-derived MSCs are derived from human bone marrow.

[0016] In some embodiments, the therapeutic MSC secretome composition further comprises one or more of the following proteins: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulatory protein, PF4, MIF, periostealin, furin, TIMP-1, trabeculin glycan, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin glycan, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20R β, SREC-II, JAM-C, TNFRI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephricin, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF R α, Selectin, Serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Coagulation regulator protein, Endoglycosides, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostrin, Flintase, TIMP-1, PAPP-A, Glycerin proteoglycan, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lutein proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinal protein, ENPP-2, CD97, CTACK, Integrin α1, EXTL3, IL-18BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte Glycol-B3, FABP6, CHI3L1, FCRLS, TFF3, Nephlebotomycin, DPPII, cIAP-1, PDGF Rb, Pentanecoprotein 3, Angiotensinogen, Follicle-stimulating hormone-inhibin factor, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactone, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral Lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLP R, LAMP1, TROY, VCAM-1, Salivary lectin-11, S100A1, PAR1, Thyroid peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM Rβ, thromboretin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1(CD56), Granulase B, Lipocontin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, conglomerate A4, brain signaling protein 4D, ROBO2, PDX-1, APRIL, neuronal rank protein, transmembrane protein-2 with ring structure, EMMPRIN, activating protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGFR1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, Pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesenchymal protein-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGFR3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MISRII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 Rα, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator protein RIIA, ANG-2, Cochlin, progerin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease protein C, cysteine ​​protease protein D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactochonin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galactochonin-9, vWF-A2, TACE, Activator protein 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, phosphatidylinositol polysaccharide 1, LAP(TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22Rα1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, P1GF-2, neurogranulin, HE4, IL-23R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-α, PGRP-S, SDF-1α, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1RII, neurofeltin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granzyme, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein 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 comprise one or more of 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-m iR-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, hs a-miR-320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-mi R-376a-3p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-mi R-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3 p, 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 or hsa-miR-99b-5p. ,

[0018] In some respects, this document discloses the use of the compositions in treating amyotrophic lateral sclerosis (ALS) in subjects of need, the compositions being produced by any of the methods described herein.

[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 advanced ALS. In some embodiments, the subject presents with limb-related symptoms. In some embodiments, the subject presents with dysphagia or speech difficulties. In some embodiments, the treatment delays the progression of ALS.

[0020] In some embodiments, the object carries one or more amino acid variations of the SOD1 protein. In some embodiments, the one or more amino acid variations include G93A. In some embodiments, the object carries one or more dipeptide repeats of 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 a human being. In some embodiments, the composition is administered intravenously to the subject.

[0022] In some implementations, the subjects showed an increase of at least about 0.1 points per month in their ALSFRS-R score after application, or a decrease of less than about 3.0 points per month, compared to their ALSFRS-R score measured before application. In some implementations, the subjects showed an increase of 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 in their ALSFRS-R score after application, compared to their ALSFRS-R score measured before application. In some embodiments, the subjects showed a monthly decrease in ALSFRS-R score of 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 after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subjects had a history of a monthly decrease in ALSFRS-R score of approximately 3.0 points prior to administration of the therapeutic MSC secretome composition.

[0023] In some embodiments, the therapeutic MSC secretome composition is administered to the subject at a dose of 0.7 to 7 million cells / kg of cell equivalents. In some embodiments, the therapeutic MSC secretome composition comprises 4 x 10 10 Up to 10x10 10 Cells / ml. In some embodiments, the therapeutic MSC secretome composition contains 5 x 10 cells / ml. 11 Up to 1.5x10 12 One extracellular vesicle. In some embodiments, the composition is applied monthly for two or more months, or once every 1, 2, or 3 months or more.

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

[0025] In some implementations, the subjects showed an increase of at least about 0.1 points per month in their ALSFRS-R score after application, or a decrease of less than about 3.0 points per month, compared to their ALSFRS-R score measured before application. In some implementations, the subjects showed an increase of 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 in their ALSFRS-R score after application, compared to their ALSFRS-R score measured before application. In some embodiments, the subjects showed a monthly decrease in ALSFRS-R score of 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 after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subjects had a history of a monthly decrease in ALSFRS-R score of approximately 3.0 points prior to administration of the therapeutic MSC secretome composition.

[0026] In some respects, this document discloses the use of compositions for the treatment of amyotrophic lateral sclerosis (ALS) in subjects of need, said compositions comprising therapeutic mesenchymal stem cell (MSC) secretome compositions comprising extracellular vesicles, wherein, after administration, said subjects have an increase of at least about 0.1 points per month in ALSFRS-R scores or a decrease of less than about 3.0 points per month in ALSFRS-R scores compared to ALS Functional Rating Scale-Revised (ALSFRS-R) scores measured before administration.

[0027] In some implementations, the subjects showed an increase in their ALSFRS-R score 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 application, compared to the ALSFRS-R score measured before application. In some embodiments, the subjects showed a monthly decrease in ALSFRS-R score of 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 after administration, compared to the ALSFRS-R score measured before administration. In some embodiments, the subjects had a history of a monthly decrease in ALSFRS-R score of approximately 3.0 points 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 further comprises one or more of the following proteins: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulator protein, PF4, MIF, periosteal protein, furin, TIMP-1, trabeculin glycan, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin glycan, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20 Rβ, SREC-II, JAM-C, TNF. RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephricin, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF R α, Selectin, Serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Coagulation regulator protein, Endoglycosides, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostrin, Flintase, TIMP-1, PAPP-A, Glycerin proteoglycan, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lutein proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinal protein, ENPP-2, CD97, CTACK, Integrin α1, EXTL3, IL-18Bpa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte Glycol-B3, FABP6, CHI3L1, FCRLS, TFF3, Neurosphingomyelin, DPPII, cIAP-1, PDGF Rb, Pentanecoprotein 3, Angiotensinogen, Follicle-stimulating hormone inhibin, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactone, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral Lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLP R, LAMP1, TROY, VCAM-1, Salivary lectin-11, S100A1, PAR1, Thyroid peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM Rβ, thromboretin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1(CD56), Granulase B, Lipocontin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, conglomerate A4, brain signaling protein 4D, ROBO2, PDX-1, APRIL, neuronal rank protein, transmembrane protein-2 with ring structure, EMMPRIN, activating protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGFR1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, Pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesenchymal protein-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGFR3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MISRII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 Rα, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator protein RIIA, ANG-2, Cochlin, progerin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease protein C, cysteine ​​protease protein D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactochonin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galactochonin-9, vWF-A2, TACE, Activator protein 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, phosphatidylinositol polysaccharide 1, LAP(TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, Csa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22Rα1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, P1GF-2, neurogranulin, HE4, IL-23R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-α, PGRP-S, SDF-1α, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1RII, neurofeltin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granzyme, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein 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 comprise one or more of 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-m iR-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, hs a-miR-320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-mi R-376a-3p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-mi R-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3 p, 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 or hsa-miR-99b-5p. ,

[0030] In some embodiments, the composition is produced as follows: (a) culturing bone marrow-derived MSCs under the following conditions to produce MSC conditioned medium: (i) oxygen tension below 5%; and (ii) a medium with a pH below 7; (b) harvesting the MSC conditioned medium; and (c) preparing 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).

[0031] In some embodiments, the culture medium is serum-free. In some embodiments, the culture medium has a glucose concentration of 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 advanced ALS. In some embodiments, the subject presents with limb-related symptoms. In some embodiments, the subject presents with dysphagia or speech difficulties. In some embodiments, the treatment delays the progression of ALS.

[0033] In some embodiments, the object carries one or more amino acid variations of the SOD1 protein. In some embodiments, the one or more amino acid variations include G93A. In some embodiments, the object carries one or more dipeptide repeats of 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 implementations, the object is a human being. In some implementations, the 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 secretion group composition administered to the subject is a cell equivalent dose of 0.7 to 7 million cells / kg. In some embodiments, the therapeutic MSC secretion group composition comprises 4 x 10 10 Up to 10x10 10 Cells / ml. In some embodiments, the therapeutic MSC secretome composition contains 5 x 10 cells / ml. 11 Up to 1.5x10 12 One extracellular vesicle. In some embodiments, the composition is applied monthly for two or more months, or once every 1, 2, or 3 months or more. Incorporation

[0036] Every patent, publication, and non-patent document cited in this application is incorporated herein by reference in its entirety, as if each were individually incorporated by reference. If the scope of a publication or patent or patent application incorporated by reference contradicts the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. Attached Figure Description

[0037] The features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments (which utilize the principles of the disclosure) and the accompanying drawings (also referred to herein as “Figures”): Figure 1AThe differences in the original ALSFRS-R scores among all subjects at each measurement time period are shown. Figure 1B The fitted linear regression analysis for each subject over time is shown to illustrate disease progression during the study period. Detailed Implementation

[0038] I. Definition As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” and “the” include plural references. It should also be noted that, unless otherwise expressly stated, the term “or” is generally used in its meaning, including “and / or.” The terms “and / or” and “any combination thereof” and their grammatical equivalents, as used herein, are interchangeable. These terms can express any combination as specifically considered. For illustrative purposes only, the phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C.” Unless the context specifically indicates a disjunctive use, the term “or” can be used in parallel or disjunctive forms.

[0039] The term "about" or "approximately" can refer to an acceptable range of error for a particular value, which can depend in part on how the value was measured or determined, such as limitations of the measurement system. For example, "about" can refer to a standard deviation of less than or greater than 1. Alternatively, "about" can refer to 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 methods, the term can refer to within orders of magnitude, within 5 times, or within 2 times the value. When a particular value is described in the application and claims, unless otherwise stated, the term "about" should be presumed to refer to an acceptable range of error for the particular value.

[0040] Throughout this application, numerical features are presented in range format. It should be understood that the range format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of any embodiment. Therefore, unless the context explicitly specifies otherwise, a range description should be understood to have all possible subranges of the specific disclosure and individual numerical values ​​of one-tenth of the lower limit unit within that range. For example, a range such as 1 to 6 should be understood to have specific disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numerical values ​​within that range, such as 1.1, 2, 2.3, 5, and 5.9. This applies regardless of how broad the range. The upper and lower limits of these intermediate ranges may be independently included in the smaller ranges and are also covered within this disclosure, subject to the explicit exclusion limits stated in the stated range. When a stated range includes one or two limits, the range excluding one or both of its included limits is also included in this disclosure, unless the context explicitly specifies otherwise.

[0041] As used in this specification and claims, the terms “comprising” (and any form of “comprising”, such as “comprise”, “comprises”), “having” (and any form of “having”, such as “having”, “suffering from”), “including” (and any form of “including”, such as “includes”, “include”), or “containing” (and any form of “containing”, such as “contains”, “contain”) are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0042] The terms "some embodiments," "implementation," "a method of implementation," or "other embodiments" used in this specification refer to specific features, structures, or characteristics described in relation to an embodiment that are included in at least some embodiments of this disclosure, but not necessarily in all embodiments. To aid in understanding this disclosure, several terms and phrases are defined below.

[0043] Certain explicit details in this description are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the invention can be practiced without these details. In other instances, well-known techniques and methods are not shown or described in detail to avoid unnecessary obscurity in the description of embodiments. Unless the context otherwise requires, throughout this specification and the following claims, the word “comprising” and variations thereof (such as “comprises”, “comprising”) are understood to have an open, inclusive meaning, i.e., “including but not limited to”. Furthermore, the headings provided herein are for convenience only and do not constitute an explanation of the scope or meaning of the claimed disclosure.

[0044] Suitable methods and materials are described below, but methods and materials similar to or equivalent to those described herein may be used in practice or experimentation with this disclosure.

[0045] II. Therapeutic Composition The components used to prepare the disclosed compositions and the compositions themselves used in the methods disclosed herein are disclosed. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, while specific references to every various individual and collective combination and arrangement of these compounds may not be explicitly disclosed, each case is specifically considered and described herein. For example, if a particular MSC secretome (including, but not limited to, MSC exosomes (with or without growth factors) referred to herein as extracellular vesicle isolates (EVIPs)) is disclosed and discussed, and various modifications that can be made to a variety of molecules including the MSC secretome are discussed, each and every combination and arrangement of the MSC secretome and possible modifications are specifically considered unless explicitly indicated to the contrary. Thus, if examples of a class of molecules A, B, and C and a class of molecules D, E, and F, and the combination molecule AD are disclosed, then each is considered individually and collectively, even if not listed individually, meaning that combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered disclosed. Similarly, any subsets or combinations of these are also disclosed. Thus, for example, subgroups of AE, BF, and CE would be considered disclosed. This concept applies to all aspects of this application, including but not limited to steps in methods of manufacturing and using the disclosed compositions. Therefore, if various additional steps can be performed, it should be understood that each of these additional steps can be performed using any particular embodiment or combination of embodiments of the disclosed methods.

[0046] The primary nutritional characteristic of MSCs is the secretion of growth factors and exosomes to induce cell proliferation and angiogenesis. Exosomes express mitogenic proteins such as transforming growth factor-α (TGF-α), TGFβ, hepatocyte growth factor (HGF), epithelial growth factor (EGF), basic fibroblast growth factor-2 (FGF-2), and insulin-like growth factor-1 (IGF-1). These increase 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 lineage cells and initiate angiogenesis. MSCs assist the regenerative environment 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-γ (INF-γ), MSCs secrete a range of growth factors and anti-inflammatory proteins with complex feedback mechanisms across many types of immune cells. Key immunomodulatory cytokines include prostaglandin 2, TGF-131, HGF, SDF-1, nitrous oxide, indoleamine 2,3-dioxygenase, IL-4, IL-10, IL-1 receptor antagonists, and soluble tumor necrosis factor-α receptors. MSCs inhibit the proliferation and function of many inflammatory immune cells, including T cells, natural killer cells, B cells, monocytes, macrophages, and dendritic cells. Although MSCs can regulate T cell activity across species, the mechanisms differ across mammalian species.

[0047] The chronic inflammatory environment is characterized by a persistent imbalance between helper T cell and macrophage types. MSC exosomes indirectly promote the conversion of TH1 cells to TH2 cells by reducing INF-γ and increasing IL-4 and IL-10. The 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 have anti-diabetic effects. Similarly, the reduction of INF-γ and the secretion of IL-4 promote the conversion of macrophages from M1 type (pro-inflammatory, anti-angiogenic, and tissue growth inhibitory) to M2 type (anti-inflammatory, pro-remodeling, and tissue healing), effects required for the healing and regeneration of bone, muscle, and nerves.

[0048] This document discloses complex compositions of secreted biomolecules (proteins, lipids, and ribonucleic acid) and / or extracellular vesicles containing biomolecules derived from mesenchymal lineage cells. In one aspect, this document discloses compositions comprising therapeutically effective amounts of MSC secretions (e.g., including but not limited to MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles containing the composition) and one or more biomolecules (e.g., peptides, polypeptides, proteins, siRNA, shRNA, and / or microRNAs (miRNAs)).

[0049] In some embodiments, the therapeutic composition comprises 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 the following proteins, any combination of the following proteins, or all of the following proteins: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF R α, Selectin, Serine protease inhibitor B6, Dkk-3, Coagulation regulator protein, PF4, MIF, Periostrin, Flin protease, TIMP-1, Gynostemma pentahydrate, PCK1, CD99, CD63, CD9, CD81, Transferrin, DcR3, Lutein glycan, TIMP-2, SLITRK5, FAP, Neurosphingomyelin, DPPII, cIAP-1, Pentanein 3, Lactobacillus, Neutral lysozyme, Albumin, Galactoglobulin-1, UNC5H3, IL-20, β-β, SREC-II, JAM-C, TNF-α, htPAPP-A, eNOS, MSP, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephric proteoglycan, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF Rα, Selectin, Serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, Coagulation regulator protein, Endoglobulin, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostrin, Flintase, TIMP-1, PAPP-A, Glycerin proteoglycan, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lutein proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinal cord protein, ENPP-2, CD97, CTACK, Integrin α1, EXTL3, IL-18, BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte-B3, FABP6, CHI3L1, FCRLS, TFF3, Neurosphingomyelin, DPPII, cIAP-1, PDGF Rb, Pentanecoprotein 3, Angiotensinogen, Follicle-stimulating hormone-inhibin factor, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactobacillus, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLPR, LAMP1, TROY, VCAM-1, salivary agglutinin-11, S100A1, PAR1, thyroid peroxidase, aminopeptidase P2, IL-1 RI, ADAMS, OSM R β, platelet-reactive protein-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), granzyme B, lipoconazole, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, conglomerate A4, brain signaling protein 4D, ROBO2, PDX-1, APRIL, neuronal rank protein, transmembrane protein-2 with ring structure, EMMPRIN, activating protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGFR1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, Pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesenchymal protein-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGFR3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MIS RII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 R α, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator RIIA, ANG-2, Cochlin, presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease C, cysteine ​​protease D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactochonin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galactochonin-9, vWF-A2, TACE, Activator protein 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, phosphatidylinositol polysaccharide 1, LAP(TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22Rα1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, P1GF-2, neurogranulin, HE4, IL-23R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-α, PGRP-S, SDF-1α, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1RII, neurofeltin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granzyme, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, and IL-13.

[0050] Extracellular vesicles (EVs) are membrane-bound microspheres containing proteins and RNA (exosomes are a subset of them). Exosomes are small (e.g., 20–150 nm) diameter lipid bilayer vesicles secreted by cells to initiate paracrine communication. Other EV populations originate directly from the plasma membrane or form during apoptosis (apoptotic bodies). This document discloses compositions containing therapeutically effective amounts of MSC secretory components (e.g., including but not limited to MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles containing the composition). In some embodiments, the therapeutic composition comprises nucleic acids 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, and 95. Or 100 or more of the following nucleic acids, any combination of the following nucleic acids, or all of 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-mi R-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-1 6-5p, 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-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 and hsa-miR-99b-5p.

[0051] Exemplary microRNA inclusions 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 a binding site for TMPRSS2 in the miRNA.

[0052] In some embodiments, the therapeutic composition comprises CD63 + CD9 - CD81 - Extracellular vesicles of the phenotype. 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. - .

[0053] In some embodiments, the MSCs cultured to produce the therapeutic composition are capable of undergoing in vitro differentiation into three lineages: adipocytes, osteoblasts, and chondrocytes. In some embodiments, the MSCs are positive for CD73, CD105, CD166, and CD90, and negative for CD14, CD31, CD34, and CD45.

[0054] It is understood and envisioned herein that the MSC secretome comprises exosomes and growth factors. The growth factors and exosomes can be heterologous or homologous. The growth factors and exosomes can be derived from cells within any human body, such as ectodermal cells, endoderm cells, or mesodermal cells. For example, the MSC secretome may comprise growth factors derived from mesenchymal stem cells (MSCs), exosomes derived from MSCs, or both growth factors and exosomes derived from MSCs. In some embodiments, the method further includes adding at least one additive containing exosomes and growth factors. Specifically, MSCs under appropriate wound healing conditions can produce suitable therapeutic agents, such as exosomes and growth factors, which can provide treatment for inflammatory lung diseases. In one aspect, this article discloses compositions in which the MSC secretome composition further comprises prostaglandin E2 (PGE2), transforming growth factor 131 (TGF-131), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), an IL-1 receptor antagonist, and soluble TNF-α receptor, insulin-like growth factor, and fibroblast growth factor (FG). F) 1-23 (especially FGF1 and FGF2), bone morphogenetic protein (BMP) 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-forming stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), estrogen and / or thyroid hormone.

[0055] Embodiments of the therapeutic compositions described herein may include proteins and microRNAs, some of which may be embedded in or surrounded by a lipid membrane to generate vesicles ranging in size from about 20 nm to about 200 nm. The number of vesicles in the composition may be about 1 million to about 100 billion vesicles per mL when suspended, or about 10 million to about 1 trillion when formulated as a lyophilized powder.

[0056] A. Delivery of drug carriers / drug products The therapeutic compositions described herein can be administered in vivo in pharmaceutically acceptable carriers. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject together with a nucleic acid or carrier without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition containing it. Carriers are naturally chosen to minimize any degradation of the active ingredient and any adverse side effects on the subject, as is well known to those skilled in the art. The compositions can be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally, in vitro, topically, etc., including top-nasal administration or inhalation. As used herein, "top-nasal administration" means delivery of the composition into the nose and nasal passages through one or both nostrils and may include delivery via a spray or droplet mechanism, or via atomization of a nucleic acid or carrier.

[0057] Inhalation of the composition may be achieved through the nose or mouth via a spray or droplet mechanism, such as a dosing inhaler, dry powder inhaler, nebulizer, vaporization device, etc. It may also be delivered directly to any area of ​​the respiratory system (e.g., the lungs) via intubation. The exact amount of composition required will vary depending on the individual, including their species, age, weight and general condition, the severity of the condition being treated, the method of administration, etc.

[0058] If the composition is used for parenteral administration, it is typically characterized by injection. Injectable formulations can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in a liquid prior to injection, or emulsions. Parenteral administration may involve the use of sustained-release or continuous-release systems to maintain a constant dose.

[0059] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th edition) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and glucose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained-release formulations, such as a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, which is in the form of a molded article, such as a membrane, liposome, or microparticle. It will be apparent to those skilled in the art that certain carriers may be preferred depending on, for example, the route of administration and the concentration of the composition applied.

[0060] Drug carriers are known to those skilled in the art. These are most typically standard carriers for human administration, including solutions such as sterile water, saline, and buffered solutions at 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.

[0061] In addition to the selected molecules, the pharmaceutical composition may also include a carrier, thickener, diluent, buffer, preservative, surfactant, etc. The pharmaceutical composition may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

[0062] The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be local (including ocular, vaginal, rectal, and intranasal), oral, by inhalation, or parenteral, such as by intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarily, or percutaneously.

[0063] 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 / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral media include sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solutions, or non-volatile oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as Ringer's glucose-based supplements), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. Preparations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily bases, thickeners, etc., may be necessary or desired.

[0064] Compositions for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants, or binders may be desired.

[0065] Some compositions may be applied in the form of pharmaceutically acceptable acid addition salts or base addition salts, which are formed by reacting with inorganic acids (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanate, sulfuric acid, and phosphoric acid) and 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 reacting with inorganic bases (such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide) and organic bases (such as monoalkylamines, dialkylamines, trialkylamines, arylamines, and substituted ethanolamines).

[0066] B. Therapeutic uses Effective dosages and administration schedules for the composition can be determined empirically, and such determinations are within the scope of the art. The dosage range used to administer the composition is sufficiently broad to produce the desired effect where the symptoms of the condition are affected. Dosages should not be too high to avoid adverse side effects such as unwanted cross-reactions, allergic reactions, etc. Typically, dosages will vary depending on the patient's age, condition, sex, and severity of the disease, as well as the route of administration or whether other medications are included in the regimen, and can be determined by a person skilled in the art. If any contraindications are present, the individual physician can adjust the dosage. Dosages can be varied and can be administered once or multiple times daily for one or several days, and / or once or multiple times monthly for two or more months, or once every one, two, or three months or more. Guidelines for appropriate dosages of given classes of medicines can be found in the literature.

[0067] C. A combination of mesenchymal stem cells and therapeutic secretory groups The therapeutic compositions disclosed herein utilize MSC secretomes and / or growth factors derived from mesenchymal stem cells (MSCs). In one aspect, this document discloses MSC secretome compositions (including, but not limited to, MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles containing the compositions). The therapeutic compositions can be used for the treatment, inhibition, reduction, amelioration, and / or prevention of conditions such as, for example, amyotrophic lateral sclerosis (ALS).

[0068] MSCs are pluripotent cells capable of differentiating into multiple cell types, including myocytes, chondrocytes, adipocytes, and osteoblasts. These cells are typically found in the placenta, umbilical cord blood, adipose tissue, bone marrow, or amniotic fluid, including perivascular tissue. As used herein, “MSC” refers to non-terminally differentiated cells, including but not limited to pluripotent stem cells, pluripotent stromal cells, stromal vascular cells, pericytes, vascular pericytes, stromal cells, pluripotent cells, fibroblast-like cells derived from adipose tissue, stromal vascular fractions derived from adipose tissue, MSCs derived from bone marrow, fibroblast-like cells derived from bone marrow, stromal vascular fractions derived from bone marrow, MSCs derived from bone marrow, fibroblast-like cells derived from tissue, adult stem cells, adult stromal cells, keratinocytes, and / or melanocytes.

[0069] In addition to their differentiation potential, MSCs possess immunomodulatory capabilities that lead to the expression of many different cytokines and growth factors. As used herein, "MSC formulation" or "MSC secretome composition" refers to a composition comprising MSC growth factors, MSC exosomes, extracellular vesicles, extracellular vesicle isolates (EVIPs), or 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 from horses, cattle, pigs, sheep, non-human primates, dogs, cats, rabbits, rats, and mice). In embodiments, the MSCs may be derived from the patient to whom the composition will be applied (autologous) or from another individual (allogeneic). MSCs may be cultured and expanded to collect conditioned medium or to increase the cell number of lysates, or used fresh before incorporation into the compositions disclosed herein. MSC secretome compositions (including, but not limited to, MSC growth factors, MSC exosomes, MSC extracts, and / or compositions comprising extracellular vesicles) may contain about 0.00001 to about 20 wt.%, such as about 0.01 to about 10 wt.%, of mesenchymal stem cell (MSC) extracts, MSC exosomes, or MSC growth factor formulations. The MSC formulation may contain MSC conditioned medium or MSC lysates from cell culture-expanded MSCs. In some embodiments, the composition may further contain about 0.01 to about 10 wt.%, of cell-free medium conditioned for the growth of MSCs or MSC lineages, wherein the cells are cultured under normal hyperoxia conditions or under artificial wound healing conditions.

[0070] As disclosed herein, MSCs used to produce the disclosed MSC additives (including frozen or powdered additives of growth factor secretome compositions) can be selectively stimulated to produce MSC growth factors, secretome, cytokines, chemokines, mesenchymal stem cell proteins, peptides, glycosaminoglycans, extracellular matrix (ECM), proteoglycans, secretome, and exosomes. Growth factors and exosomes can be derived from any cells in the human body, such as ectoderm cells, endoderm cells, or mesodermal cells. As used herein, MSC growth factors include, but are not limited to, prostaglandin E2 (PGE2), transforming growth factor 131 (TGF-1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonists and soluble TNF-α receptors, insulin-like growth factor, and fibroblast growth factor (FGF) 1-23 (…). In particular, FGF1 and FGF2), bone morphogenetic protein (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-a (TGF-a), 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 hormones including estrogen and thyroid hormone.

[0071] MSC culture can occur under wound healing and / or hypoxic conditions. Hypoxic conditions can include approximately 1% to approximately 5% oxygen, reduced or absent serum, reduced glucose, or various combinations of these elements. This combination of reduced nutrients and metabolites can trigger cultured cells to produce wound-healing and anti-inflammatory ECM proteins and growth factors to guide tissue healing. This guidance of tissue healing may occur in the form of novel ECM proteins, such as collagen and glycosaminoglycans (GAGs), as well as growth factors and cytokines. In one aspect, MSC formulations (e.g., MSC secretome compositions) comprise MSC growth factors, MSC exosomes, and / or MSC cell extracts or MSC lysates obtained from MSCs cultured under standard hyperoxia conditions (e.g., 21% oxygen) or under artificial wound healing conditions (e.g., 0.1% to approximately 5% oxygen).

[0072] As disclosed herein, artificial wound healing conditions mimic the growth conditions in real wounds, where reduced nutrient supply and waste removal are typically caused by local blood circulation disruptions. This creates a harsh environment for cells until new blood vessels are formed and blood circulation is restored. Therefore, artificial wound healing conditions for culturing MSCs can include one or more of the following growth conditions: reduced glucose availability, reduced oxygen tension, decreased pH, and increased temperature.

[0073] In some embodiments, glucose availability may be reduced relative to a normal control (e.g., 4.5 g / L). The glucose in modified culture media that reduce glucose without harming cells may be reduced by 0% to 50%, more preferably by about 5% to 40%. For example, MSC artificial wound healing culture conditions may include glucose reductions of about 5% to about 15%, about 10% to about 20%, about 15% to about 25%, about 20% to about 30%, or about 25% to about 35%. In some embodiments, glucose is present at a concentration of about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 g / L, or at a concentration in the range of any two of these values. In some embodiments, glucose is present at a concentration of less than or no more than 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.

[0074] In some implementations, oxygen tension can be reduced to the oxygen levels of hypoxic conditions. Normal atmospheric oxygen is approximately 21%, and any reduction is considered hypoxic. Therefore, in one aspect, MSCs can be cultured at oxygen levels of 0.0% to 20.9%, from about 0.1% to about 0.5%, from about 0.1% to about 2.0%, from about 0.1% to about 5.0%, from about 0.5% to 5.0%, from about 1.0% to about 10%, from about 5.0% to about 10.0%, and from about 10.0% to about 15.0%. Hypoxic conditions can be one aspect of artificial wound healing conditions.When culturing MSCs to produce a therapeutic secretory composition containing extracellular vesicles and / or growth factors secreted by MSCs, the oxygen tension can be between about 0.5% and 20.5% oxygen, for example, such as 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, 10.6, 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, 15.7, 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, or 20.0% oxygen, or a range between any two of these values.

[0075] The pH value can also be lowered during MSC culture. The pH can be from about 6.0 to about 7.4, for example, from about 6.0 to about 6.4, from about 6.2 to about 6.4, from about 6.2 to about 6.6, from about 6.4 to about 6.6, from about 6.4 to about 6.8, or from about 6.6 to about 7.0, such as 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.

[0076] The temperature of the culture environment can be increased relative to the physiological homeostatic temperature (e.g., 37°C). In one aspect, the culture conditions for MSCs can include from about 35°C to about 39°C, from about 35°C to about 36°C, from about 36°C to about 37°C, from about 37°C to about 38°C, from about 38°C to about 39°C, and from about 39°C to about 40°C. In another aspect, the culture temperature can 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, 3... 7.4, 37.5, 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℃.

[0077] 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 any range between two of these values. In some embodiments, the culture medium contains from 8% to 12%, 5% to 15%, or 9% to 11% HPL by volume.

[0078] In one aspect, MSC secretome compositions (including, but not limited to, MSC growth factors, MSC exosomes, MSC extracts, and / or compositions containing extracellular vesicles) may further include a protective coating (e.g., cryoprotectant oligosaccharides and protein solutions) to reduce the degradation of growth factors. It is understood and contemplated herein that the protective coating may be engineered as a polymer. “Polymer” refers to a relatively high molecular weight organic compound, natural or synthetic, whose structure can be represented by repeating small unit monomers. Non-limiting examples of polymers include polyethylene, rubber, and cellulose. Synthetic polymers are typically formed by the addition or condensation polymerization of monomers. The term “copolymer” refers to a polymer formed from two or more distinct repeating units (monomer residues). For example, but not limited to, copolymers can be alternating copolymers, random copolymers, block copolymers, or graft copolymers. It is also contemplated that, in some aspects, each block of a block copolymer may itself contain a copolymer. The term “polymer” encompasses all forms of polymers, including but not limited to natural polymers, synthetic polymers, homopolymers, heteropolymers, or copolymers, addition polymers, etc. In one aspect, the gel matrix may include copolymers, block copolymers, diblock copolymers, and / or triblock copolymers. In one aspect, the protective coating may include a biocompatible polymer. In another aspect, the biocompatible polymer may be cross-linked. Such polymers may also be used to slowly release fat browning agents and / or fat modifiers into tissues. Biocompatible polymers as used herein include, but are not limited to, polysaccharides; hydrophilic peptides; polyamino acids such as poly-L-glutamic acid (PGS), γ-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 polyethylene oxide (PEO); poly(oxyethylated polyols); polyenols; polyvinylpyrrolidone; poly(hydroxyalkyl methacrylamide); poly(hydroxyalkyl methacrylate); polysaccharides; poly(hydroxy acids); poly(vinyl alcohol); polyhydroxy acids such as poly(lactic acid), polyglycolic acid, and polylactic-glycolic acid copolymers; and polyhydroxyalkanoic acids. Esters such as poly(3-hydroxybutyrate) or poly(4-hydroxybutyrate); polycaprolactone; poly(orthoester); polyanhydride; poly(phosphazene); polylactide-caprolactone copolymer; polycarbonates such as tyrosine polycarbonate; polyamides (including synthetic and natural polyamides), peptides and poly(amino acids); polyesteramide; polyester; poly(p-dioxanone); poly(alkylene oxide); hydrophobic polyether; polyurethane; polyether ester; polyacetal; polycyanoacrylate; polyacrylate; polymethyl methacrylate; polysiloxane; poly(ethylene oxide) / poly(propylene oxide) copolymer; polyketide; polyphosphate; polyhydroxyvalerate; polyalkylene oxalate; polyalkylene succinate; poly(maleic acid) and its copolymers.Biocompatible polymers may also include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohol (PVA), methacrylate PVA (m-PVA), polyethylene ethers, polyethylene esters, polyhalogenated polyethylene, polyvinylpyrrolidone, polyglycolic acid, polysiloxanes, polyurethanes and their copolymers, alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylates and methacrylates, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethylcellulose, cellulose triacetate, sodium cellulose sulfate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(methyl methacrylate), poly(methyl methacrylate) The following polymers are included: isobutyl acrylate, 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 blends thereof. Exemplary biodegradable polymers include polyesters, poly(orthoesters), polyvinylamine, poly(caprolactone), poly(hydroxybutyrate), poly(hydroxyvalerate), polyanhydride, poly(acrylic acid), polyglycolic acid, poly(glycolic acid), poly(urethane), polycarbonate, polyphosphate, polyphosphazene, their derivatives, their linear and branched copolymers and block copolymers, and mixtures thereof.

[0079] In some embodiments, the protective coating comprises a carbohydrate structure of monosaccharides and carbohydrate polymers (such as disaccharides or polysaccharides, including but not limited to non-reducing polysaccharides or disaccharides and any combination thereof). Examples of carbohydrates that can be used in the protective coating include glucose, aldoses (D-allose, D-agrose, D-mannose, etc.), glucopyranose, pentahydroxyhexanal, aD-glucopyranosyl-D-glucose, aD-glucopyranosyl-dihydrate, polymers of PD-glucopyranosyl units, PD-fructofuranosyl-aD-glucopyranoside (anhydrous / dihydrate), f3-D-galactopyranosyl-D-glucose, aD-glucopyranosyl-dihydrate, etc. Glycosyl-aD-glucopyranoside (anhydrous / dihydrate), galactose, pentose (ribose, xylose, lysose), dextrose, decahydrate, fructose, sucrose, lactose, maltose, trehalose, agarose, D-galactosyl-O-(1-4)-dehydrated-L-galactosyl, cellulose, polymers of PD-glucopyranoside units and starch, as well as polyols, polyols, sugar alcohols, erythritol, glycols, glycerol, xylitol and sorbitol.

[0080] In some embodiments, the protective coating contains a biocompatible and / or biodegradable polyester or polyanhydride, such as poly(lactic acid), poly(glycolic acid), and polylactic-glycolic acid copolymers. The particles may comprise one or more of the following: homopolymers comprising glycolic acid units (referred to herein as “PGA”), and lactic acid units (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, collectively referred to herein as “PLA”), and caprolactone units (such as poly(e-caprolactone) (collectively referred to herein as “PCL”); and copolymers comprising lactic acid and glycolic acid units, such as various forms of polylactic-glycolic acid copolymers and polylactide-glycolic acid copolymers, characterized by a lactic acid:glycolic acid ratio, collectively referred to herein as “PLGA”; and polyacrylates and their derivatives. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers". In some embodiments, the PEG region can be covalently associated with the polymer via a pyrolytic linker to create a "PEGylated polymer". In one aspect, the polymer comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% acetal side groups.

[0081] The triblock copolymers disclosed herein include, for example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), polyvinylpyrrolidone-vinyl acetate copolymer, polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic-glycolic acid), polylactic-glycolic acid copolymer (PLGA), and cellulose derivatives such as hydroxymethyl cellulose and hydroxypropyl cellulose. Examples of diblock copolymers that can be used in the protective coatings disclosed herein include, for example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), polyvinylpyrrolidone-vinyl acetate copolymer, polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic-glycolic acid), polylactic-glycolic acid copolymer (PLGA).

[0082] In one aspect, the protective coating comprises (i.e., encapsulated), and the encapsulated composition may further comprise lecithin or hydrolyzed lecithin as a carrier or as an encapsulating material. As used herein, lecithin and / or hydrolyzed lecithin coatings comprise coatings containing phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid. The source of lecithin may be plant or animal.

[0083] In one aspect, any of the polymers, monosaccharides, disaccharides, or polysaccharides used to form the protective coating formed by placing the MSC additive in the encapsulation solution can be at an appropriate concentration for forming the protective coating. For example, the polymers, monosaccharides, disaccharides, or polysaccharides can be at any concentration between 0.01 mM and 10.0 M, such as from about 0.01 M to about 0.1 M, from about 0.1 mM to about 1.0 M, or from about 1.0 M to about 10.0 M.

[0084] In one aspect, the MSC secretome compositions disclosed herein (including, but not limited to, MSC growth factors, MSC exosomes, MSC extracts, and / or extracellular vesicles comprising the compositions) may include any known ingredients commonly found in the pharmaceutical field, such as agents for combating free radicals; bactericides; chelating agents; preservatives; alkalizing 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 ingredients 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-ascorbic acid, magnesium ascorbate-2-phosphate, ascorbate palmitate, tetrahexyldecyl ascorbate), vitamin E (tocotrienols), vitamin A (retinol, retinal, retinoic acid, provitamin A carotenoids, such as β-carotene), N-acetylglucosamine, or other derivatives of glucosamine. 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-linolenic acid (GLA), alpha-linolenic acid (ALA), di-homo-gamma-linolenic acid (DGLA), and arachidonic acid (ARA). Fatty acids can be derived from a variety of 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 derivatives of glucosamine, ultra-low molecular weight (ULMW) hyaluronic acid, chondroitin sulfate, or keratin sulfate.

[0085] The production of MSC secretome compositions may include culturing MSCs collected from donors to generate a culture medium under culture conditions, in some embodiments including reduced oxygen and nutrients; stimulating cultured cells to selectively secrete desired anti-inflammatory proteins, peptides, glycosaminoglycans, proteoglycan exosomes, and secretomes by modulating cell growth conditions; collecting, combining, and freezing the aggregate mixture with an encapsulation solution, wherein the aggregate mixture comprises exosomes, peptides, proteins, cytokines, growth factors, extracellular matrix (ECM), proteoglycans, glycosaminoglycans; and chemokines selected from human MSCs, animal MSCs, pluripotent stromal cells, and fibroblasts; combining the aggregate mixture with an encapsulation solution (such as an oligosaccharide, such as a trehalose solution or a protein solution) and freezing the mixture; and lyophilizing or freeze-drying the frozen mixture to produce a dry powder. Alternatively, MSCs can be lysed to collect all MSCs from the culture process, generating extracted lysates; the extracted lysates can be concentrated and combined with an encapsulation solution (such as oligosaccharides, like trehalose solution or protein solution), and the mixture can be frozen; and the frozen mixture can be lyophilized or freeze-dried to generate a dry powder. The powder contains a highly concentrated collection of analgesic MSC secretomes and exosomes, as well as extracellular matrix components specific for anti-inflammatory effects.

[0086] The method also includes filtration-sterilization, concentration, freezing, or freeze-drying of MSC conditioned media. Furthermore, MSC media can be combined with cryoprotectants and then frozen.

[0087] There are various methods for lysing MSCs. Lysis can be achieved by adding hypotonic solutions or by repeated freeze-thaw cycles that disrupt the cell membrane. Alternatively, cells can lyse while attached to a culture surface or in suspension. Cells can also be lysed via enzymatic release and / or mechanical homogenization.

[0088] MSCs can be stimulated to selectively secrete desired anti-inflammatory proteins, peptides, glycosaminoglycans, proteoglycans, exosomes, and secretomes by adjusting cell growth conditions such as cell confluence, culture medium additives, nutrient additives, oxygen levels, culture duration under these conditions, cell passage number, or combinations thereof.

[0089] III. Treatment methods for amyotrophic lateral sclerosis (ALS) In some embodiments, the therapeutic compositions disclosed herein are used in a method of treating a subject with amyotrophic lateral sclerosis (ALS). Any of the therapeutic compositions described herein may be used in this method. ALS is a complex, progressive, and fatal neurodegenerative disease that affects motor neurons; the death (atrophy) of motor neurons over time leads to muscle weakness, loss of muscle mass, and inability to control movement, and ultimately death due to respiratory failure.

[0090] In some implementations, the subject has spinal-onset ALS. In some implementations, the subject has medullary-onset ALS. The subject may have advanced ALS. The subject may have limb-related symptoms. The subject may have dysphagia or speech difficulties.

[0091] In some embodiments, treatment using the therapeutic compositions disclosed herein delays the progression of ALS.

[0092] In some implementations, gene mutations can be associated with ALS. In one example, a mutation in superoxide dismutase type 1 (SOD1) has been found in ALS patients. In another example, a six-nucleotide repeat amplification (GGGGCC(G4C2)) of the six nucleotides in the C9ORF72 gene is considered to be associated with familial ALS. In some implementations, individuals with ALS may carry mutations in either the SOD1 or C9ORF72 gene, which can result in amino acid variations in the SOD1 or C9ORF72 protein, respectively. In some implementations, individuals with ALS may carry one or more amino acid variations in the SOD1 gene. For example, an individual with ALS may carry one or more amino acid variants, including K3E, A4V, W32*, G38R, G41S, G72S, N86S, D90A, G93A, S105L, D109Y, C111Y, I112M, L126*, N139D, L144S, or combinations thereof, where * indicates a truncation introduced due to an early stop codon in the SOD1 protein. In some embodiments, an individual with ALS may carry G4C2 amplification in the C9ORF72 gene, resulting in a longer form of the C9ORF72 protein with dipeptide repeats. Non-restrictive dipeptide repeats may 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).

[0093] In some implementations, a questionnaire-based scale (ALS Functional Rating Scale-Revised (ALSFRS-R)) can be used to measure and track changes in physical function in ALS patients over time. The ALSFRS-R measures 12 aspects of physical function, including language, salivation, swallowing, handwriting, food cutting, stair climbing, turning over in bed, walking, dressing and hygiene, dyspnea (difficulty breathing), orthopnea (shortness of breath when lying down), and respiratory insufficiency. Each function is scored from 4 (normal) to 0 (incapacitated), with a maximum total score of 48 and a minimum total score of 0. For example, patients with higher ALSFRS-R scores across the 12 aspects are considered to have more physical function. In some implementations, the ALSFRS-R can be conducted by a healthcare provider.

[0094] In some embodiments, subjects may experience an increase in their ALSFRS-R score after treatment with the therapeutic composition described herein. For example, subjects may experience an increase of at least about 0.1 points per month in their ALSFRS-R score compared to an ALSFRS-R score measured before treatment. In some embodiments, subjects may experience an increase of 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 in their ALSFRS-R score compared to an ALSFRS-R score measured before application. In some implementations, subjects may have an increase in their ALSFRS-R score 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 monthly application of the therapeutic composition described herein for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, compared to the ALSFRS-R score measured before application.

[0095] In some embodiments, subjects may experience a decrease in their ALSFRS-R score after treatment with the therapeutic composition described herein. For example, subjects may experience a decrease in their ALSFRS-R score by less than about 3.0 points per month compared to an ALSFRS-R score measured before treatment. In some embodiments, subjects may experience a decrease in their ALSFRS-R score by less than about 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 compared to an ALSFRS-R score measured before application. In some implementations, subjects may have a monthly decrease in ALSFRS-R score of less than about 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 compared to the ALSFRS-R score measured before application, after monthly application of the therapeutic composition described herein for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more.

[0096] In some implementations, the subject may have a history of a decline in ALSFRS-R score prior to administration of the therapeutic MSC secretome composition. For example, the subject may have a history of a monthly decline in 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.

[0097] This article also describes a method for treating ALS in subjects, including administering to the subject a composition comprising secreted extracellular vesicles containing any combination of proteins and / or miRNAs selected from the following: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulator protein, PF4, MIF, periosteal protein, furin, TIMP-1, trabeculin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20R. β, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephricin, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside-A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSF Ra, coagulation regulatory protein, endosaccharides, IGFBP-3, RGM-C, PF4, MIF, TGM4, periosteal protein, furin protease, TIMP-1, PAPP-A, dermal proteoglycan, PCK1, arylsulfatase A, CD99, CA2, PRDX4, transferrin, DcR3, GP73, LAIR2, ULBP-4, luminal proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, spinal proteinENPP-2, CD97, CTACK, integrin α1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, glyoxalase II, trypsin I, IGF-2R, ADAMTSL-1, proerythropoietin, cyclophosphamide D1, DNMT3A, BCL-2, CL-P1, hepatocyte glycoside B3, FABP6, CHI3L1, FCRLS, TFF3, leptospirin, DPPII, cIAP-1, PDGF Rb, pentacyclic protein 3, angiotensinogen, follicle-stimulating hormone inhibin, CF VII, persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, endothelin, granuloprotein precursor, PCSK2, GKN1, IL-18, neutral lysozyme, stabilizer-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLP R, LAMP1, TROY, VCAM-1, Salivary lectin-11, S100A1, PAR1, Thyroid peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM Rβ, thromboretin-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, LAMA4Lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Convolutional protein A4, Brain signaling protein 4D, ROBO2, PDX-1, APRIL, Neural rank protein, cyclic transmembrane protein-2, EMMPRIN, activator protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, Brain signaling protein 7A, NKp80, Prolactin, Cysteine ​​protease protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesentery protein-2, osteoadhesin, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α5, VAMP-2, FLRG, Ret metaphase factor, CD73, TRACP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated protein C Type I lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MIS RII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycans, neurotrophic factors, ILT4, uPAR, Axl, WIF-1IL-7 Rα, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator RIIA, ANG-2, Cochlin, progerin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease C, cysteine ​​protease D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease protein SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactokinin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFA5, Galactokinin-9, vWF-A2, TACE, Activator protein RUB, Cathepsin S, LDL R, BMPR-IAOX40, 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, phosphatidylinositol polysaccharide 1, LAP (TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-bl, FABP4, RGM-A, RELT, TrkC, C5a, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-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 α1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, PIGF-2, neurogranulin, HE4, IL-23 R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-RH, PGRP-S, SDF-1a, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1 MI, neurofeedin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granulin, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, Granulase A, 2B4, ESAM, IL-1R4, CXCL14, IL-31, SIRP α, Urinary Modulatory Protein, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3, FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Fibrin-1, Reg4, ILT2, Mer, TREM-2Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, Procalcitonin, SOX15, Kallikrein 11, BCMA, Dendritic Cell-Associated C-Lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Cohesin-Glycan-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein EM, FUT8, B7 -H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, Kallikrein 7, 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-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hs a-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-13 0a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, h sa-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-mi r-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-2 6a-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、h sa-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, hs a-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-1hsa-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. ,

[0098] In some embodiments, the therapeutic product is administered at a cell equivalent dose range of 0.7 to 7 million cells / kg. In some embodiments, the therapeutic product is administered at a cell equivalent dose of at least about, and at most about, or about 0.2, 0.5, 0.7, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12.0 million cells / kg, or any two of these values. In some embodiments, the product is administered at a dose providing 9 x 10 11 Up to 1.2x10 12 One extracellular vesicle or 5x10 11 Up to 1.5x10 12 6x10 11 Up to 1.4x10 12 7x10 11 Up to 1.3x10 12 8x10 11 Up to 1.2x10 12 Or 8x10 11 Up to 1.3x10 12 A dose of extracellular vesicles is administered. In some embodiments, the product is administered to provide at least or at most 5 x 10-1 11 6x10 11 7x10 11 8x10 11 9x10 11 1x10 12 1.1x10 12 1.2x10 12 1.3x10 12 1.4x10 12 Or 1.5x10 12 A dose of 1 extracellular vesicle is administered. In some embodiments, the therapeutic product comprises 6 x 10-1 10 Up to 8x10 10 5x10 10 Up to 9x10 10 4x10 10 Up to 10x10 10 5.5x10 10 Up to 8.5x10 10Or 6x10 10 Up to 8.5x10 10 Cells / ml. In some implementations, the therapeutic product contains 6 x 10 cells / ml. 10 Up to 8x10 10 1 extracellular vesicle or cell per milliliter and administered at a dose of 10 to 20 ml. In some embodiments, the therapeutic product contains 6 x 10 10 Up to 8x10 10 One extracellular vesicle or cell per milliliter and administered at a dose of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ml or any two of these values.

[0099] In some embodiments, the object to be treated with the therapeutic products and methods described herein may include any object suffering from or diagnosed with ALS. In some embodiments, the object may include mammals. In some embodiments, the object may include humans. In some embodiments, the object may include non-human mammals. Non-limiting examples of non-human mammals may include non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as mice, rats, and guinea pigs.

[0100] IV. Examples The following examples are provided to provide those skilled in the art with 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 and not to limit the disclosure. Efforts have been made to ensure accuracy regarding numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be taken into account. These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0101] A. Example 1 - Production of the therapeutic composition The MSC secretome therapeutic composition was prepared by the following method: Human bone marrow-derived MSCs were cultured in culture dishes with growth medium to expand the MSC population. The growth medium was then removed, and the cells were washed with PBS. The MSCs were then cultured under hypoxic conditions in a glucose-reduced medium with a pH less than 7.0. The conditioned medium was then collected and percolated, followed by sterilization. The production of the therapeutic product was carried out in accordance with Good Manufacturing Practice and Current Good Tissue Practice.

[0102] The tetraspan membrane protein profile of extracellular vesicles present in the therapeutic composition was determined, and it was found that more than 95% of the extracellular vesicles present in the therapeutic composition were CD63. + CD9 - CD81 - .

[0103] The protein contents of the therapeutic product were identified, and the following proteins were found: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulator protein, PF4, MIF, periosteal protein, furin, TIMP-1, trabeculin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20 Rβ, SREC-II, JAM-C, TNF-α, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, convolutional protein A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephrine, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1, R6 GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside-A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSF Ra, coagulation regulatory protein, endosaccharides, IGFBP-3, RGM-C, PF4, MIF, TGM4, periosteal protein, furin, TIMP-1, PAPP-A, dermal proteoglycan, PCK1, arylsulfatase A, CD99, CA2, PRDX4, transferrin, DcR3, GP73, LAIR2, ULBP-4, luminal proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, spinal cord protein, ENPP-2, CD97, CTACK, integrin α1, EXTL3, IL-18BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte Glycol-B3, FABP6, CHI3L1, FCRLS, TFF3, Nephlebotomycin, DPPII, cIAP-1, PDGF Rb, Pentanecoprotein 3, Angiotensinogen, Follicle-stimulating hormone-inhibin factor, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactone, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral Lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLP R, LAMP1, TROY, VCAM-1, Salivary lectin-11, S100A1, PAR1, Thyroid peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSMR β, thromboretin-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1(CD56), Granulase B, Lipconin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Convolutional protein A4, Brain signaling protein 4D, ROBO2, PDX-1, APRIL, Neuro-rankin, Circular transmembrane protein-2, EMMPRIN, Activator protein RIB, Neuroligand 2, Epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesrin-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MISRII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 Rα, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator protein RIIA, ANG-2, Cochlin, progerin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease protein C, cysteine ​​protease protein D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CFXIV, PYY, HGF, I-TAC, Brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Gastrin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, galactagogue-9, vWF-A2, TACE, activator protein 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, phosphatidylinositol polysaccharide 1, LAP (TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 Rα1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, PIGF-2, neurogranulin, HE4, IL-23 R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, myocardial trophoprotein-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNFRII, PGRP-S, SDF-1a, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1 RII, neurofeedin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granulin, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7, and IL-13.

[0104] The nucleic acid content of the therapeutic product was determined, and the following nucleic acids were found: 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-m iR-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, hs a-miR-320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-mi R-376a-3p, hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-mi R-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3 p, 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. ,

[0105] B. Example 2 - Pilot safety study of treatment for amyotrophic lateral sclerosis (ALS) using investigational products of extracellular vesicles derived from human bone marrow stem cells. In this embodiment, the inventors report that the investigational product (IP) of human bone marrow stem cell-derived extracellular vesicles (hBM-MSC EV) is safe and effective in patients with amyotrophic lateral sclerosis (ALS). Ten ALS patients received two 10 mL intravenous (IV) infusions of IP administered one month apart, and were evaluated over a period of three months. HBM-MSC EV demonstrated safety in ALS patients. This early study suggests the need for controlled studies of EVs for the treatment of ALS.

[0106] Amyotrophic lateral sclerosis (ALS) is a neurological disorder affecting the brain and spinal cord, leading to loss of muscle control. Currently, there is no cure for ALS, and the disease worsens over time. Potential new treatments using mesenchymal stem cell extracellular vesicles (MSC EVs) are being investigated. MSC EVs are small structures containing useful molecules and proteins that can be transported to cells affected by the disease, helping to reduce inflammation and promote repair. This 3-month study examined the safety of human bone marrow MSC-EVs (hBM-MSC EVs) as a treatment given to 10 ALS patients, and how well they could delay disease progression. The inventors found no serious side effects caused by the treatment and discovered that hBM-MSCEVs have the potential to delay ALS progression. This finding indicates that more, larger studies are needed to uncover treatment details such as dosage (how much treatment is given) and frequency (how often treatment is given), and how they relate to patient outcomes.

[0107] ALS is the third most common neurodegenerative disease, after Alzheimer's and Parkinson's, and is the most common type of motor neuron disease. Despite an incidence of approximately 0.005%, progress in the understanding and treatment of ALS has been minimal. The disease manifests in both spinal and medullary forms. Genetic causes have been identified, but these events account for a minority of cases. Less clearly defined environmental factors are therefore assumed to be the cause of 90-95% of ALS cases, and until further research elucidates other causes, these cases are considered sporadic in origin. The remaining 5-10% of cases with a clear genetic association to a family history are classified as familial, and no effective treatment targeting this specific area has been developed. Patients are diagnosed through exclusion and symptom-based assessment. Research now focuses on the loss of regulation and / or clearance of protein waste products (i.e., TDP-43, SOD1, and FUS), the role of C9orf72 hexanucleotide repeat amplification, the most commonly associated mutations, and quantifying the contribution of other genes to the disease.

[0108] Riluzole, which reduces intraneuronal glutamate levels, is defined as an oral glutamatergic neurotransmission inhibitor. It has demonstrated limited clinical efficacy, with patients surviving only an additional five to six months. Edaravone, a potent intravenously (IV) administered antioxidant, slows the progression of early-stage ALS, but recent publications have cast doubt on the results of earlier clinical trials. Currently, only two drugs are FDA-approved for the treatment of ALS, and access to these treatments is likely limited and restricted. Given these limitations, the discovery and development of alternative medical treatments to halt or reverse the progression of both sporadic and familial ALS is urgent.

[0109] ExoFloTM (The research product (IP) used in this article is an EV product derived from human BM-MSCs (hBM-MSC EV). This IP is a stable EV product with a wide range of properties, including advanced particle analysis, proteomics evaluation, and USP. <71> Aseptic properties are guaranteed. Furthermore, manufactured in a cGMP environment, this IP is a high-quality biopharmacological product, stable in terms of dosage and bioactivity. Two peer-reviewed studies have demonstrated the safety of intravenous administration of the IP to severely ill COVID-19 patients. The IP has also demonstrated potency in a subset of these severely ill patients. We hypothesize that intravenous (IV) administration of this IP to ALS patients is safe and its potency can potentially be demonstrated in this patient population.

[0110] method Approval and informed consent form. The study protocol was reviewed and approved by the Institutional Review Board (JC-ALS-001) and the Institute of Regenerative and Cellular Medicine (IRCM) (ICRM-2021-296). Based on these approvals, an open-label, two-center, single-investigator pilot safety study was conducted with ten participants.

[0111] Research design. Subjects meeting the following original selection criteria were included: aged 30–65 years, diagnosed with ALS, and with written informed consent obtained from the subject or their legal representative. IRB-approved protocol biases that extended the age limit to 72 years were documented to improve recruitment of 10 subjects. Subjects were categorized based on the initial symptoms as either spinal episodic (initially presenting with limb involvement) or medullary episodic (initially presenting with dysphagia or speech difficulties). Informed consent was obtained from all subjects. ALS type was based on the initial symptom presentation of their disease, and the rate of decline was determined as rapid or general. Spinal episodic ALS declines by an average of one point per month; however, medullary episodic ALS can decline much faster. The duration of symptom presence was defined as the time elapsed since the initial report of ALS symptoms, typically much later than the onset of any related symptoms prior to diagnosis.

[0112] Safety was defined as the absence of any adverse or serious adverse events associated with the investigation product (IP). For subject #5, whose Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) score was 1 at the start of the study, was a subjective assessment provided by the patient's caregiver. This included eye contact, eye focus, and blinking communication, as the patient was unable to move or speak.

[0113] The IP dose was calculated based on (1) a Phase I START trial using IV administration of BM-MSCs for acute respiratory distress syndrome, which demonstrated safety at upper doses of up to 5 million cells / kg and 10 million cells / kg; (2) observations of approximately 2,000 EVs secreted per cell; (3) laboratory analysis indicating 60-80 billion EVs / mL; and (4) prior safe administration of a 10 mL dose to a single ALS patient. This indicated an upper limit IV IP dose of 17.5 mL / 70 kg adult, and a 10 mL IV IP was determined to be a reasonable dose providing 0.6-0.8 trillion EV particles per dose.

[0114] Each participant underwent a baseline physical examination and assessment using the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) score. Previous scores were recorded in the participant's medical records if available, but were not included in this study. The ALSFRS-R score was recorded at the first visit (time 0) immediately before the IV administration of 10 mL of intravenous insulin (IP), and again one month later immediately before the second visit (time 1), followed by the IP infusion. In the absence of IP infusion, two additional follow-up contacts (in person or online) were conducted at one-month intervals (times 2 and 3), for a total of four visits across the three-month period (see chart). The ALSFRS-R score at each visit and any documented side effects experienced after IP administration were recorded. No respiratory trials were performed in this study. All 10 patients fully adhered to the study protocol and completed the study.

[0115] statistics. The data were subjected to a Gaussian distribution of the residuals using GraphPad Prism 9.5.1 software, followed by repeated measures one-way ANOVA with Geisser-Greenhouse correction. Simple linear regression analysis of the ALSFRS-R scores and slope calculation were also performed using Prism.

[0116] result Seven men and three women consented to treatment. No subjects received riluzole during the study. Seven subjects had spinal ALS. Based on reported function and an ALS FRS-R score that decreased by at least 3 points per month in the months immediately preceding the study, three of these spinal ALS subjects experienced rapid progression. Three subjects had medullary ALS. The age range of subjects was 39 to 72 years, with a mean of 53.7 years. The time from ALS diagnosis to study start ranged from 2 to 54 months, with a mean of 20.9 months, and half of the subjects presented with a pre-existing condition. Seven subjects were Caucasian, one was African American, and two were Asian (Indian and Armenian).

[0117] The baseline ALSFRS-R score at time 0 across all 10 subjects ranged from 1 to 41, with a mean of 26.7 (Table 1). Time 0 and time 1 scores were obtained immediately preceding the start of the first and second treatments, respectively. Scores for each progression were recorded at monthly intervals, with a total of three consecutive months of sustained scores (time 0 through time 3). One subject with a previous ALSFRS-R score of 1 was considered an outlier and was included in the study to contribute to the safety assessment across all stages of disease progression.

[0118] One-way ANOVA analysis of ALSFRS-R scores for all 10 subjects at all four time points showed no significant difference in group means at any of the four time points (F(1.236, 11.12) = 4.06, p = 0.062). Nine non-outlier subjects exhibited baseline ALSFRS-R scores ranging from 18 to 41, with a mean of 29.6. In the whole group, the ALSFRS-R score ranged from 1 to 42 on the last day of the study, with a mean of 23.6, while in the group excluding outliers, the ALSFRS-R score ranged from 13 to 42, with a mean of 26.1. Over the three-month study period, all 10 subjects experienced a mean decrease of 3.1 points, and the nine non-outlier subjects experienced a mean decrease of 3.5 points.

[0119] Figure 1A The differences in the original ALSFRS-R scores among all objects in each measurement time period are shown. Figure 1B The fitted linear regression analysis for each subject over time is shown to illustrate disease progression during the study period. One subject (S3) demonstrated stable improvement in the ALSFRS-R score, while two others (S4 and S10) showed no significant reduction in their ALSFRS-R scores. The remaining subjects exhibited a decline in their scores. The mean (+ / - SD) ΔFRS calculated from Table 1 across all 10 subjects from baseline (time 0) to the study endpoint (36 months, time 3) was -1.03 + / - 1.44. The mean slope derived from the linear regression analysis was -0.98 + / - 1.67.

[0120] Table 1 below describes the ALSFRS-R scores of the objects.

[0121] a ΔFRS is calculated as (Time 3 score - Time 0 baseline score) / 3 months.

[0122] b The slope is derived from a linear regression analysis from time 0 to time 3. discuss Patients with ALS lack effective treatment options. Novel treatments are needed to reduce mortality and maintain function. MSC-derived EVs offer a novel treatment option due to preclinical and clinical evidence of safety and efficacy. Safety is a key aspect of early-stage studies for all new investigational products in novel disease indications. In this study, ten ALS subjects were treated with hBM-MSCEV IP to evaluate safety risks and efficacy potential. No adverse or serious adverse events were associated with the investigational product. This safety profile is consistent with the excellent safety profile observed in patients with severe COVID-19 when up to two doses (15 mL each) of IP were administered intravenously. This small, open-label pilot safety study demonstrates that intravenous delivery of bone marrow-derived MSC EVs is safe in ALS patients.

[0123] Regarding the mechanisms underlying the efficacy of hBM-MSC EVs against ALS, recent studies indicate a variety of possible molecular, biochemical, and cellular mechanisms at play due to the presence of EV-carried miRNAs and proteins within and around the CNS. This multi-entity cargo can actively modulate synaptic plasticity, neurogenesis, axonal growth, glial cell function, apoptosis, immune regulation, and other responses important for nervous system function. For example, several miRNA species contained in BM-MSC EVs can modulate the inflammatory, anti-inflammatory, and neurotoxic activities of astrocytes and / or microglia isolated from SOD1G93A mice (an ALS mouse model), as well as the inflammatory, anti-inflammatory, and neurotoxic activities of motor neurons differentiated from induced neural progenitor cells from ALS patients carrying SOD1G93A or C9orf72 mutations. The net effect of BM-MSC EVs in in vitro and in vivo models is a reduction in both neurotoxicity and neuroinflammation. Furthermore, the role of MSC EVs in regulating autophagy, cytoplasmic shuttle and lysosomal flow, as well as the disruption of presynaptic vesicle dynamics due to common C9orf72 ALS mutations, suggests that specific BM-MSC EV-associated miRNAs can directly improve synaptic function.

[0124] The ability of BM-MSC EVs to cross the blood-brain barrier offers the opportunity to treat this end-stage disease using a safer IV administration route. It will also allow for more convenient and frequent dosing to help prolong and increase any potential benefits from this promising treatment option.

Claims

1. A method of treating amyotrophic lateral sclerosis (ALS) in a subject of need, the method comprising administering a composition to the subject, the 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 - .

2. The method of claim 1, wherein, compared with the ALS Functional Rating Scale Revised Version (ALSFRS-R) score measured before application, the subject has an increase of at least about 0.1 points per month or a decrease of less than about 3.0 points per month in the ALSFRS-R score after application.

3. The method of claim 2, wherein, compared with the ALSFRS-R score measured before application, the subject has an increase of 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 application.

4. The method of claim 2, wherein the subject has a monthly decrease in ALSFRS-R score of 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 after application, compared to the ALSFRS-R score measured before application.

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

6. A method of treating amyotrophic lateral sclerosis (ALS) in a subject in need, the method comprising administering to the subject a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles, wherein, after administration, the subject has an increase of at least about 0.1 points per month in the ALS Functional Rating Scale-Revised (ALSFRS-R) score or a decrease of less than about 3.0 points per month in the ALS Functional Rating Scale-Revised (ALSFRS-R) score measured before administration.

7. The method of claim 6, wherein the subject has an increase in ALSFRS-R score of 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 application, compared with the ALSFRS-R score measured before application.

8. The method of claim 6, wherein the subject has a monthly decrease in ALSFRS-R score of 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 after application, compared with the ALSFRS-R score measured before application.

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

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

11. The method of any one of claims 1 to 10, wherein the therapeutic MSC secretome composition further comprises one or more of the following proteins: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulatory protein, PF4, MIF, periosteal protein, furin, TIMP-1, trabeculin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20Rβ, SREC-II, JAM-C, TNF. RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephricin, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside-A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, coagulation regulatory protein, endosaccharides, IGFBP-3, RGM-C, PF4, MIF, TGM4, periosteal protein, furin, TIMP-1, PAPP-A, dermal proteoglycan, PCK1, arylsulfatase A, CD99, CA2, PRDX4, transferrin, DcR3, GP73, LAIR2, ULBP-4, luminal proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, spinal cord protein, ENPP-2, CD97, CTACK, integrin α1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte Glycol-B3, FABP6, CHI3L1, FCRLS, TFF3, Neurosphingomyelin, DPPII, cIAP-1, PDGFRb, Pentanecocin 3, Angiotensinogen, Follicle-stimulating hormone-inhibin factor, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactone, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral Lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLPR, LAMP1, TROY, VCAM-1, salivary agglutinin-11, S100A1, PAR1, thyroid peroxidase, aminopeptidase P2, IL-1 RI, ADAMS, OSM R β, platelet-reactive protein-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), granzyme B, lipoconazole, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, conglomerate A4, brain signaling protein 4D, ROBO2, PDX-1, APRIL, neuronal rank protein, transmembrane protein-2 with ring structure, EMMPRIN, activating protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, Pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesenchymal protein-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MIS RII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 R α, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator RIIA, ANG-2, Cochlin, presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease C, cysteine ​​protease D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactochonin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galactochonin-9, vWF-A2, TACE, Activator protein 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, phosphatidylinositol polysaccharide 1, LAP(TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-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 α1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, PIGF-2, neurogranulin, HE4, IL-23 R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-α, PGRP-S, SDF-1α, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1RII, neurofeltin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granzyme, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7 or IL-13.

12. The method of any one of claims 1 to 11, wherein the extracellular vesicles comprise one or more of 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-1 32-3p, 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-181 a-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-mi R-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-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 or hsa-miR-99b-5p., 13. The method of any one of claims 1 to 12, wherein the composition is produced as follows: (a) Culture bone marrow-derived MSCs under the following conditions to produce MSC conditioned medium: (i) oxygen tension below 5%; and (ii) Culture media with a pH below 7; (b) Harvesting the MSC conditioned medium; and (c) Prepare 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 of step (a).

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

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

16. The method of any one of claims 1 to 15, wherein the subject suffers from episodic ALS.

17. The method of any one of claims 1 to 15, wherein the subject suffers from medullary paroxysmal ALS.

18. The method of any one of claims 1 to 17, wherein the subject suffers from advanced ALS.

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

20. The method of any one of claims 1 to 19, wherein the subject exhibits difficulty swallowing or speech.

21. The method of any one of claims 1 to 20, wherein the treatment delays the progression of ALS.

22. The method of any one of claims 1 to 21, wherein the object carries one or more amino acid variations in the SOD1 protein.

23. The method of claim 22, wherein the one or more amino acid variations include G93A.

24. The method of any one of claims 1 to 21, wherein the object carries one or more dipeptide repeats of the C9ORF72 protein.

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

26. The method of any one of claims 1 to 25, wherein the object is a person.

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

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

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

30. The method of any one of claims 1 to 28, wherein the therapeutic MSC secretome composition comprises 4 x 10 10 Up to 10x10 10 Cells / ml 31. The method of any one of claims 1 to 28, wherein the therapeutic MSC secretome composition comprises 5 x 10 11 Up to 1.5x10 12 One extracellular vesicle.

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

33. A method of preparing a composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition for treating amyotrophic lateral sclerosis (ALS) in subjects of need, the method comprising: (a) Culture bone marrow-derived MSCs under the following conditions to produce MSC conditioned medium: (i) Oxygen tension below 5%; as well as (ii) Culture media with a pH below 7; (b) Harvesting the MSC conditioned medium; and (c) Prepare 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 of step (a).

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

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

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

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

38. The method of any one of claims 33 to 37, wherein the therapeutic MSC secretome composition comprises one or more of the following proteins: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulator protein, PF4, MIF, periosteal protein, furin, TIMP-1, trabeculin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20Rβ, SREC-II, JAM-C, TNF. RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephricin, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1, R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside-A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, coagulation regulatory protein, endosaccharides, IGFBP-3, RGM-C, PF4, MIF, TGM4, periosteal protein, furin, TIMP-1, PAPP-A, dermal proteoglycan, PCK1, arylsulfatase A, CD99, CA2, PRDX4, transferrin, DCR3, GP73, LAIR2, ULBP-4, luminal proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, spinal cord protein, ENPP-2, CD97, CTACK, integrin α1, EXTL3, IL-18, BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte-B3, FABP6, CHI3L1, FCRLS, TFF3, Neurosphingomyelin, DPPII, cIAP-1, PDGF Rb, Pentanecoprotein 3, Angiotensinogen, Follicle-stimulating hormone-inhibin factor, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactobacillus, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLPR, LAMP1, TROY, VCAM-1, salivary agglutinin-11, S100A1, PAR1, thyroid peroxidase, aminopeptidase P2, IL-1 RI, ADAMS, OSM R β, platelet-reactive protein-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), granzyme B, lipoconazole, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, conglomerate A4, brain signaling protein 4D, ROBO2, PDX-1, APRIL, neuronal rank protein, transmembrane protein-2 with ring structure, EMMPRIN, activating protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGFR1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, Pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesenchymal protein-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGFR3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MIS RII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 R α, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator RIIA, ANG-2, Cochlin, presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease C, cysteine ​​protease D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactochonin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galactochonin-9, vWF-A2, TACE, Activator protein 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, phosphatidylinositol polysaccharide 1, LAP(TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22Rα1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, P1GF-2, neurogranulin, HE4, IL-23R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-α, PGRP-S, SDF-1α, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1RII, neurofeltin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granzyme, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7 or IL-13.

39. The method of any one of claims 33 to 38, wherein the therapeutic MSC secretome composition comprises one or more of 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, hs a-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-148 a-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, hs a-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, h sa-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-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 or hsa-miR-99b-5p.

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

41. The use as claimed in claim 40, wherein the subject suffers from episodic ALS.

42. The use as claimed in claim 40, wherein the subject suffers from medullary paroxysmal ALS.

43. The use as claimed in any one of claims 40 to 42, wherein the subject suffers from advanced ALS.

44. The use as claimed in any one of claims 40 to 43, wherein the object exhibits limb-related symptoms.

45. The use as claimed in any one of claims 40 to 44, wherein the subject exhibits difficulty swallowing or speech difficulties.

46. ​​The use as described in any one of claims 40 to 45, wherein the treatment delays the progression of ALS.

47. The use as claimed in any one of claims 40 to 46, wherein the object carries one or more amino acid variations in the SOD1 protein.

48. The use as described in claim 47, wherein the one or more amino acid variations include G93A.

49. The use as claimed in any one of claims 40 to 46, wherein the object carries one or more dipeptide repeats of the C9ORF72 protein.

50. The use as claimed in claim 49, wherein the one or more dipeptide repeats comprise poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

51. The use as claimed in any one of claims 40 to 50, wherein the object is a person.

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

53. The use as described in claim 52, wherein, compared with the ALS Functional Rating Scale Revision (ALSFRS-R) score measured before application, the subject has an increase of at least about 0.1 points per month or a decrease of less than about 3.0 points per month in the ALSFRS-R score after application.

54. The use as claimed in claim 53, wherein the subject has an increase in ALSFRS-R score of 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 application, compared with the ALSFRS-R score measured before application.

55. The use as claimed in claim 53, wherein the subject has a monthly decrease in ALSFRS-R score of 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 after application, compared to the ALSFRS-R score measured before application.

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

57. The use as claimed in any one of claims 52 to 56, wherein the dose of the therapeutic MSC secretome composition administered to the subject is a cell equivalent dose of 0.7 to 7 million cells / kg.

58. The use according to any one of claims 52 to 56, wherein the therapeutic MSC secretome composition comprises 4 x 10 10 Up to 10x10 10 Cells / ml 59. The use according to any one of claims 52 to 56, wherein the therapeutic MSC secretome composition comprises 5 x 10 11 Up to 1.5x10 12 One extracellular vesicle.

60. The use as described in any one of claims 52 to 58, wherein the composition is applied monthly for two or more months, or once every one, two, or three months or more.

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

62. The use as claimed in claim 61, wherein the composition is administered intravenously to the subject.

63. The use as described in claim 62, wherein, compared with the ALS Functional Rating Scale Revision (ALSFRS-R) score measured before application, the subject has an increase of at least about 0.1 points per month or a decrease of less than about 3.0 points per month in the ALSFRS-R score after application.

64. The use as claimed in claim 63, wherein the subject has an increase in ALSFRS-R score of 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 application, compared with the ALSFRS-R score measured before application.

65. The use as claimed in claim 63, wherein the subject has a monthly decrease in ALSFRS-R score of 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 after application, compared to the ALSFRS-R score measured before application.

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

67. Use of the composition for the treatment of amyotrophic lateral sclerosis (ALS) in subjects of need, said composition comprising a therapeutic mesenchymal stem cell (MSC) secretome composition containing extracellular vesicles, wherein, after administration, said subjects have an increase of at least about 0.1 points per month in their ALS Functional Rating Scale-Revised (ALSFRS-R) score or a decrease of less than about 3.0 points per month in their ALS Functional Rating Scale-Revised (ALSFRS-R) score compared to a score measured before administration.

68. The use as claimed in claim 67, wherein the subject has an increase in ALSFRS-R score of 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 application, compared with the ALSFRS-R score measured before application.

69. The use as claimed in claim 67, wherein the subject has a monthly decrease in ALSFRS-R score of 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 after application, compared to the ALSFRS-R score measured before application.

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

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

72. The use according to any one of claims 61 to 71, wherein the therapeutic MSC secretome composition further comprises one or more of the following proteins: ferritin, NUP85, LAMP2, GPR115, serine protease inhibitor F1, OPN, PAI-1, DAPP1, cathepsin B, brain signaling protein 6C, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, coagulation regulatory protein, PF4, MIF, periostealin, furin, TIMP-1, trabeculin, PCK1, CD99, CD63, CD9, CD81, transferrin, DcR3, clavicin, TIMP-2, SLITRK5, FAP, leptospirin, DPPII, cIAP-1, pentacyclic protein 3, endothelin, neutral lysozyme, albumin, galactoglobulin-1, UNC5H3, IL-20Rβ, SREC-II, JAM-C, TNF. RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 α, ST6GAL1, CD99-L2, conglomerate A4, EMMPRIN, p53, brain signaling protein 7A, NKp80, cysteine ​​protease protein B, osteoadhesion, mesonephricin, calreticulin, osteoactivin, asparagine endopeptidase, TAZ, cathepsin L, RBP4, serine protease inhibitor A4, JAM-A, MCSF, LIMPII, OPG, IL-22, galactagogue-3, MOG, trypsin 3, SIRP α, and cohesin-glycan-4, and at least one protein selected from the following: ferritin, IGFBP-4, IL-1R6, GSTM1, NUP85, LAMP2, transmembrane peptidase A, IL-1 F10, bIG-H3, GPR115, TGFbI, liver glycoside-A4, CD109, serine protease inhibitor F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, brain signaling protein 6C, IGF-2, PDGF Rα, selectin, serine protease inhibitor B6, Dkk-3, CNTF, TSP-1, GM-CSFRa, coagulation regulatory protein, endosaccharides, IGFBP-3, RGM-C, PF4, MIF, TGM4, periosteal protein, furin, TIMP-1, PAPP-A, dermal proteoglycan, PCK1, arylsulfatase A, CD99, CA2, PRDX4, transferrin, DcR3, GP73, LAIR2, ULBP-4, luminal proteoglycan, TIMP-2, TFPI, SOX2, SLITRK5, FAP, spinal cord protein, ENPP-2, CD97, CTACK, integrin α1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20Ra, Glyoxalase II, Trypsin I, IGF-2R, ADAMTS L1-1, Pro-erythropoietin, Convolutional D1, DNMT3A, BCL-2, CL-P1, Hepatocyte Glycol-B3, FABP6, CHI3L1, FCRLS, TFF3, Neurosphingomyelin, DPPII, cIAP-1, PDGFRb, Pentanecocin 3, Angiotensinogen, Follicle-stimulating hormone-inhibin factor, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Lactone, Granulosin precursor, PCSK2, GKN1, IL-18, Neutral Lysozyme, Stabilin-2, IL-17 RD, albumin, follicle-stimulating hormone-inhibin-like protein 1, MMP-10, FKBP51, LRRC4, Pref-1, galactagogue-1, troponin C, UNC5H3, FLRT2, CD314, brain signaling protein 6B, cytokinin-4, CD27 ligand, IL-20 Rβ, brain signaling protein 6A, TSK, cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, norin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Glycopropyl peptide, Activator Protein 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, Phosphatidylinositol Glycan 2, MSP R, DSCAM, Proteolytic enzyme, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nestin-1, CD2, Kallikrein 1, TSLPR, LAMP1, TROY, VCAM-1, salivary agglutinin-11, S100A1, PAR1, thyroid peroxidase, aminopeptidase P2, IL-1 RI, ADAMS, OSM R β, platelet-reactive protein-2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), granzyme B, lipoconazole, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, lipoprotein, AIF, SorCS2, SULT2A1, CD39L2, insulin R, HIF-1 α, OX40 ligand, Pax3, UCH-L3, cMASP3, Langerin, desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, conglomerate A4, brain signaling protein 4D, ROBO2, PDX-1, APRIL, neuronal rank protein, transmembrane protein-2 with ring structure, EMMPRIN, activating protein RIB, neuroligand 2, epithelial regulatory protein, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, extracellular matrix protein-2, sFRP-3, p53, EphB3, NCK1, brain signaling protein 7A, NKp80, prolactin, cysteine ​​agonist protein B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Brain signaling protein 6D, FGF-3, GATA-4, VAP-A, CHST2, Pregnancy-associated plasma protein-2, cohesin-3, serrated protein 1, AKR1C4, olfactory mesenchymal protein-2, osteoadhesion, NKp44, thyroglobulin, IL-21R, chemokines, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, integrin α 5. VAMP-2, FLRG, Ret metaphase factor, CD73, TRAP, proGRP, granzyme H, PRX2, p27, salivary lectin-6, dendritic cell-associated C-type lectin-1, CD51, Notch-1, calreticulin, DR3, DCTN1, CDC25B, bone activator, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, testosterone glycan 2, phosphatidylinositol glycan 5, CD6, salivary lectin-2, asparagine endopeptidase, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1β, TAFA1, podocyte protein, RalA, CRELD2, GRAP2, SP-D, BID, GFR α-2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, cathepsin L, 6-Ckine, MIS RII, kallikrein 5, TGM3, FCAR, contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, disaccharide proteoglycan, neurotrophic factor, ILT4, uPAR, Axl, WIF-1, IL-7 R α, GPR56, CEACAM-3, MCEMP1, FABP2, plexin B3, MEPE, activator RIIA, ANG-2, Cochlin, presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, stalkin-1, GUSB, nestin-2, IL-17F, SR-AI, TAFA2, N-cadherin, IL-17B, IL-17RC, MIP-3b, cysteine ​​protease C, cysteine ​​protease D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, intestinal peptidase, cathepsin E, TSLP, TCN2, GDF-15, epidermal morphogenetics, GRKS, PD-1, serine protease inhibitor A4, ADAM23, NOV, galactagogue-2, neuronal surface protein 3β, TLR3, Sirtuin 2, Numb, IL-28 Rα, IL-33, Lin28, FCRL1, KLF4, NKp30, lymphocyte chemokine, cysteine ​​protease inhibitor SN, JAM-A, calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, kallikrein 14, extracellular matrix protein-3, Oligo2, kallikrein 12, CA13, IL-9, stalk protein-3, MPIF-1, cysteine ​​protease protein S, ADA, IL-2 Rb, GFR α-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, transmembrane serine 1 protease, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, brain signaling protein 4C, SorCS3, Tie-1, IL-31RA, Arginase 1, POGLUT1, IL-1ra, Flatfoot protein, TIM-3, CREG, CD300f, uPA, EphA2, LLRTM4, LIMPII, Tenosynovin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Salivary lectin-9, ICAM-3, Cysteine ​​protease inhibitor SA, Galactochonin-4, Pepsinogen II, Desmosome core protein-3, Stalk protein-4, SCF, Serine protease inhibitor A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFAS, Galactochonin-9, vWF-A2, TACE, Activator protein 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, phosphatidylinositol polysaccharide 1, LAP(TGFb1), Gash, contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-α, NRG1-131, FABP4, RGM-A, RELT, TrkC, CSa, SREC-I, neural epithelial stem cell protein, TPO, ErbB3, Kirrel3, FLRT1, galactagogue-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 α1, AMIGO2, HCC-1, TFPI-2, ULBP-2, desmosome core protein 2, agglutinin, synaptic fusion protein 4, VAMP-1, stalkin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, PIGF-2, neurogranulin, HE4, IL-23 R, galactagogue-7, GALNT3, GITR L, CD14, R-reactive protein 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF-α, PGRP-S, SDF-1α, PDGF-AB, GPVI, CD40, SCF R, thromboretin-5, IL-1RII, neurofeltin-2, cadherin-13, E-selectin, GITR, WISP-1, renin, AgRP, MDL-1, ROBO3, RANTES, endothelial cell-specific molecules, granzyme, hCGb, mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, trypsin 3, ARSB, LIF Rα, BAFF R, CD157, granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL-31, SIRP α, urinary modulatory proteins, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 α, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, fibrinogen-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Synaptic Fusion Protein 6, GRO, Bcl-w, Lipocrine-2, PDGF-AA, IL-2 Ra, Angiopoietin, LYVE-1, CD4, RAGE, CDNF, Short Proteoglycan, NAP-2, PU.1, EDAR, ADAMTS13, Kynurenase, PTH1R, IFN-γ R1, CrkL, B7-1, PARC, Draxin, VE-cadherin, procalcitonin, SOX15, kallikrein 11, BCMA, dendritic cell-associated C-lectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, resistin, HVEM, ENPP-7, cohesin-4, IL-2 Rg, MICA, dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, glycoprotein V, brain signaling protein 4G, IL-12p40, total PSA, IL-15, MAP1D, Clq, TNF4, Dtk, endothelial glycoprotein, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, galactagogue-8, CA4, cysteine ​​protease protein EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7 or IL-13.

73. The use according to any one of claims 61 to 72, wherein the extracellular vesicle comprises one or more of 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-1 32-3p, 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-181 a-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-mi R-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-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 or hsa-miR-99b-5p.

74. The use according to any one of claims 61 to 73, wherein the composition is produced as follows: (a) Culture bone marrow-derived MSCs under the following conditions to produce MSC conditioned medium: (i) oxygen tension below 5%; and (ii) Culture media with a pH below 7; (b) Harvesting the MSC conditioned medium; and (c) Prepare 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 of step (a).

75. The use as described in claim 74, wherein the culture medium is serum-free.

76. The use as described in claim 74 or 75, wherein the culture medium has a glucose concentration of less than 4.5 g / L.

77. The use as claimed in any one of claims 61 to 76, wherein the subject suffers from episodic ALS.

78. The use as claimed in any one of claims 61 to 76, wherein the subject suffers from medullary paroxysmal ALS.

79. The use as claimed in any one of claims 61 to 78, wherein the subject suffers from advanced ALS.

80. The use as claimed in any one of claims 61 to 79, wherein the object exhibits limb-related symptoms.

81. The use as claimed in any one of claims 61 to 80, wherein the subject exhibits difficulty swallowing or speech difficulties.

82. The use as claimed in any one of claims 61 to 81, wherein the treatment delays the progression of ALS.

83. The use as described in any one of claims 61 to 82, wherein the object carries one or more amino acid variations in the SOD1 protein.

84. The use as described in claim 83, wherein the one or more amino acid variations include G93A.

85. The use as claimed in any one of claims 61 to 82, wherein the object carries one or more dipeptide repeats of the C9ORF72 protein.

86. The use as described in claim 85, wherein the one or more dipeptide repeats comprise poly-GA, poly-GP, poly-GR, poly-PA, or poly-PR.

87. The use as claimed in any one of claims 61 to 86, wherein the object is a person.

88. The use as claimed in any one of claims 74 to 87, wherein the bone marrow-derived MSC is derived from human bone marrow.

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

90. The use as claimed in any one of claims 61 to 89, wherein the dose of the therapeutic MSC secretory group composition administered to the subject is a cell equivalent dose of 0.7 to 7 million cells / kg.

91. The use according to any one of claims 61 to 89, wherein the therapeutic MSC secretome composition comprises 4 x 10 10 Up to 10x10 10 Cells / ml 92. The use according to any one of claims 61 to 89, wherein the therapeutic MSC secretome composition comprises 5 x 10 11 Up to 1.5x10 12 One extracellular vesicle.

93. The use as described in any one of claims 61 to 92, wherein the composition is applied to the subject monthly for two or more months, or once every one, two, or three months or more.

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