Methods of treating amyotrophic lateral sclerosis

Abstract

Described herein are neonatal cardiac mesenchymal progenitor cells, an nMPC secretome, or an nMPC exosome for treating Amyotrophic Lateral Sclerosis (ALS) in a subject. One or more doses of neonatal cardiac mesenchymal progenitor cells, an nMPC secretome, an nMPC exosome, an nMPC total secretome, or a combination thereof can be administered to the subject. Neonatal cardiac mesenchymal progenitor cells can be allogeneic. The subject may also be administered one or more combination therapies.

Description

WSGR Ref. No. : 64320-709.601METHODS OF TREATING AMYOTROPHIC LATERAL SCLEROSISCROSS REFERENCE

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 769,522, filed March 10, 2025, and U. S. Provisional Application No. 63 / 712,683, filed October 28, 2024, which applications are incorporated herein by reference in their entireties.BACKGROUND

[0002] Amyotrophic Lateral Sclerosis (ALS), or Lou Gehrig's disease, is a neurodegenerative disease characterized by motor neuron loss in the primary motor cortex, the brainstem, and the spinal cord, resulting in generalized muscle weakness and atrophy. Progressive deterioration of motor function in patients severely impairs critical physiological functions such as breathing and swallowing, and the disease typically causes death within about 2 to 5 years after diagnosis. Neuroinflammatory processes are implicated in the progression of ALS and represent a facet of the disease that may be targeted. There is an unmet need for novel and effective therapies to treat patients with Amyotrophic Lateral Sclerosis.SUMMARY OF THE DISCLOSURE

[0003] Described herein, in certain aspects, are compositions comprising neonatal cardiac mesenchymal progenitor cells (nMPCs) and uses thereof in the treatment of cardiomyopathy in subjects with Amyotrophic Lateral Sclerosis (ALS). Described herein, in certain aspects, are compositions comprising nMPC secretomes (conditioned medium), nMPC exosomes, an nMPC total secretome (total conditioned medium), or a combination thereof in the treatment of cardiomyopathy in subjects with Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the ALS is familial ALS.

[0004] Described herein, in some aspects, is a method of treating Amyotrophic Lateral Sclerosis (ALS) in a subject, comprising administering to the subject neonatal cardiac mesenchymal progenitor cells (nMPCs), an nMPC secretome (conditioned medium), nMPC exosomes, an nMPC total secretome (total conditioned medium), or any combination thereof, whereby ALS is treated. In certain embodiments, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM- 1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4. In some embodiments, the subject is non-ambulatory. In other embodiments, the subject is ambulatory. In one non-limiting embodiment, nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4. In some embodiments, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2. In some instances, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, or SOX2. In other instances, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2. In some embodiments, the nMPCs are further negative for protein expression of p!6INK4aup to at least passage 8. In one instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, or SOX2 and (b)WSGR Ref. No. : 64320-709.601 negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, or p!6INK4a. In some embodiments, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In another instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In some embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, or 8 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, andNkx2.5; (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4; (c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / or (d) negative for protein expression of p!6INK4aup to at least passage 8. In some embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. In some embodiments, the nMPCs are genetically engineered. In some embodiments, the nMPCs are genetically engineered to overexpress one or more paracrine factors. In some embodiments, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. In some embodiments, the nMPCs are genetically engineered to over express HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1. In some embodiments, the nMPCs are non-naturally occurring. In some embodiments, the nMPCs are not xenogenic. In some embodiments, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5, (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4, and are genetically engineered to express or overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. Alternatively, or in addition, nMPCs can be further negative for protein expression of pl6INK4aup to at least passage 8. In one instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a, and (c) genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1.

[0005] In certain embodiments, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4. In some embodiments, the subject is non-ambulatory. In other embodiments, the subject is ambulatory. In one non-limiting embodiment, nMPCs are (a) positive for cell surface protein expression of c-kit (CD 117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5 and (b) negative for protein expression of CD34,WSGR Ref. No. : 64320-709.601CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4. In some embodiments, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2. In some instances, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, or SOX2. In other instances, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2. In some embodiments, the nMPCs are further negative for protein expression of p!6INK4aup to at least passage 8. In one instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, or SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, or p!6INK4a. In some embodiments, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and p!6INK4a. In another instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and p!6INK4a. In some embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, or 8 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5; (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4; (c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / or (d) negative for protein expression of p!6INK4aup to at least passage 8. In some embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. In some embodiments, the nMPCs are genetically engineered. In some embodiments, the nMPCs are genetically engineered to overexpress one or more paracrine factors. In some embodiments, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. In some embodiments, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1. In some embodiments, the nMPCs are non-naturally occurring. In some embodiments, the nMPCs are not xenogenic. In some embodiments, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5, (b) negative forprotein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4, and are genetically engineered to express or overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. Alternatively, or in addition, nMPCs can be further negative for protein expression of pl 6INK4aup to at least passage 8. In one instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3,WSGR Ref. No.: 64320-709.601SSEA4, and pl6INK4a, and (c) genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1.

[0006] Alternatively, or in addition, described herein is a method of treating ALS in a subject in need thereof, comprising administering to the subject an nMPC secretome (conditioned medium) or nMPC exosomes. In some embodiments, an nMPC secretome is an nMPC total secretome (total conditioned medium) comprising both an nMPC secretome and nMPC exosomes. In some embodiments, the nMPC secretome (e.g, a “nMPC secretome” or “nMPC conditioned medium”) or exosomes are positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and / or Akt. In some embodiments, the nMPC secretome comprises HSF1 at levels higher than HSF1 in the secretome of aMPCs. In some embodiments, the secretome or exosomes are negative for protein expression of IGF1. In one instance, the secretome is positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and / or Akt. Alternatively, or in addition, in certain embodiments, the secretome is positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of FUS, TAF15, CAP2, SNX9, PSMB7, SUMO1, EIF3A, ECHI, HNRNPL, NTM, ANG, ERP44, NAA38, RRM2B, RAB1A, RAB1B, PGRMC1, ERH, TIGAR, ITGA2, SGTA, AR1C1, AKR1C2, DNASE2, HSP90AB4P, ANGPTL2, SF3B2, GREM1, ARF3, ARF1, ARF5, LSM5, PSMD9, SUGT1, ALDH1A3, PDGFRB, HIST1H1C, SP100, G6PD, PPIL1, SNRPD1, FDPS, RAB6B, RAB6A, DCTD, GLB1, ARIH1, IST1, HNRNPU, ULBP2, RAET1G, ERAP2, CDC42, EDF1, LIPG, FKBP4, RBMX, RBMXL1, RPL30, THY1, AP2M1, RPS16, FHL3, BZW1, BZW2, EIF4G1, PFDN4, CLTB, PIR, MVP, ECU, DCI, ALYREF, RPS10, PPP3CA, TAX1BP3, PPA1, DDX39B, DDX39A, GNPNAT1, LIMA1, SNRPD3, ADH5, ENAH, DYNLL1, DYNLL2, HUWE1, HMGB2, PTMS, UCHL3, PFDN2, DYNLRB1, HDGFRP2, INF2, HPRT1, EIF2S2, ADAMT7, EIF3C, EIF3CL, RPS5, VPS35, CS, ECHS1, UBQLN1, GBP2, GBP1, SF3B1, RANBP1, GNS, HLA-B, UBXN1, GNPDA1, IGF2, EIF3B, TCEB1, VASP, GSR, HLA-A, HLA-HIGF2, NUDT5, COPE, HNRNPA3, SRI, HNRNPC, MMP10, EWSR1, IPO7, YARS, TMPO, HNRNPH1, HLA-A, PDGFC, IL1B, S100A10, TXNDC2, DPY30, LAMP1, LYPLA1, CBR1, ARHGAP1, TPR, NAMPT, NAMPTL, CXCL2, EIF4A2, RPS4X, PABPN1, PHLDB1, NAP1L4, DDX6, PSMC3, MAP7D1, OSCAR, TES, NMT1, NMT2, MAT2A, PRMT1, COX17, SELM, CARS, PPP2CA, PPP2CB, VEGFC, VPS26A, PXN, PAWR, STRAP, RAB11A, RABI IB, EIF5, COPZ1, COPS3, CHMP4B, PDXK, RALA, RALB, SEC13, FHL2, TOMI, PYCARD, PDLIM4, RBBP4, RBBP7, RPS20, SRSF1, DLD, SNRPE, EIF4EBP1, RPLPO, RPLP0P6, LSM3, SLC16A3, CSE1L, NUCKS1, TSNAX, RPSA, RPSAP58, AP1B1, EIF2S1, LCP1, PSMD4, AARS, SRSF2, SFRS2, TRIP10, CLTA, USO1, VBP1, UBE2K, MRPL12, UAP1, HIST1H1B, YBX3, RPL10A, IDH1, SNRPN, SNRPB, PCK2, EIF6, MMP3, DHX9, MATR3, UBE2D2, UBE2D3, PTRF, ACTR1A, LAMA1, PDCD6IP, QARS, HN1L, PLBD2, OS9, ILF3, ISOCI, PPP1R7, MAPK1, PDCD1LG2, GOLGB1, HSPA9, ACBD3, BTF3, UBE2I, SRRT, EIF4H, FERMT2, EIF3G, PARVA, DBNL, BOLA2, MAPRE1, PRDX5, STX12, EIF1, SMS, GRPEL1, OAF, SYNCRIP, ECE1, VAPA, PCNP, PSME1, EIF3J, C I4orf 166, DDX1, AKR1B1, SRP9, PCMT1, XPO1, MMP9, RPS7, NRCAM, FKBP3, SSB, DSC3, XRCC5, CCT6A, LIF, SERPINA9, SND1, ANP32B, G3BP1, GPC6, GLOD4,WSGR Ref. No.: 64320-709.601RRBP1, TGFB1, USP14, PLAUR, CXCL3, FASN, C0PB2, TARDBP, TNFAIP6, RPS3, NDUFAB1, LMNB1, C0L16A1, PSMF1, PHGDH, PRKCDBP, CXCL5, YAP1, TR0VE2, MACF1, EIF5B, STX7, PFDNS, AP0A1, PDAP1, STX12, EIF3K, FH, RBM8A, EIF1, RPS2, PTGR1, EEA1, CAB39, DCTN2, GDF15, MYL9, TCP1, RPS12, RAD23A, SKP1, PDLIM1, HSPH1, PSMB3, SUB1, API5, TWF2, KTN1, DYNC1H1, SNRNP70, MCFD2, PPP1R18, PTGES3, APEX1, TNKS1BP1, FUBP1, VAT1, PAFAH1B1, CBX1, PIK7, HNRPDL. ZNF185, DYNC1I2, KHDRBS1, SFPQ, PDCD5, TRIM28.PPIC, NARS, PCBP2, CD2AP, PSMA1, CAPRIN1, KHSRP, NSFL1C, PPP1R12A, NUDC, MFAP2, IGF2R, RGMB, PABPC1, MAN1A1, DDB1, PTBP1, FBN2, CNBP, OTUB1, NASP, HSPD1, EEF1D, RNPEP, CRKL, HNRNPAB, EIF3F, PAICS, ILF2, COPB1, PA2G4, COP A, NT5E, HMGA1, SF1, XRCC6, GNB2L1, MEI, MTAP, CCT2, ARCN1, PEPD, and EPB41L3. Alternatively, or in addition, in certain embodiments, the secretome is positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of PPP1CA, PPP1CC, STI 3. ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8-MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP, NAP1L1, HSP90AA1, SET, EN01, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, 42254, and PAFAH1B2. In one instance, the secretome does not comprise 42254. Alternatively, or in addition, in certain embodiments, a secretome is positive for one, two, three, four, five, six, seven, eight, nine, ten, or more of FUS, TAF15, CAP2, SNX9, PSMB7, SUM01, EIF3A, ECHI, HNRNPL, NTM, ANG, ERP44, NAA38, RRM2B, RAB1A, RAB1B, PGRMC1, ERH, TIGAR, ITGA2, SGTA, AR1C1, AKR1C2, DNASE2, HSP90AB4P, ANGPTL2, SF3B2, GREM1, ARF3, ARF1, ARF5, LSM5, PSMD9, SUGT1, ALDH1A3, PDGFRB, HIST1H1C, SP1OO, G6PD, PPIL1, SNRPD1, FDPS, RAB6B, RAB6A, DCTD, GLB1, ARIH1, IST1, HNRNPU, ULBP2, RAET1G, ERAP2, CDC42, EDF1, LIPG, FKBP4, RBMX, RBMXL1, RPL30, THY1, AP2M1, RPS16, FHL3, BZW1, BZW2, EIF4G1, PFDN4, CLTB, PIR, MVP, ECU, DCI, ALYREF, RPS10, PPP3CA, TAX1BP3, PPA1, DDX39B, DDX39A, GNPNAT1, LIMA1, SNRPD3, ADH5, ENAH, DYNLL1, DYNLL2, HUWE1, HMGB2, PTMS, UCHL3, PFDN2, DYNLRB1, HDGFRP2, INF2, HPRT1, EIF2S2, ADAMT7, EIF3C, EIF3CL, RPS5, VPS35, CS, ECHS1, UBQLN1, GBP2, GBP1, SF3B1, RANBP1, GNS, HLA-B, UBXN1, GNPDA1, IGF2, EIF3B, TCEB1, VASP, GSR, HLA-A, HLA-HIGF2, NUDT5, COPE, HNRNPA3, SRI, HNRNPC, MMP10, EWSR1, IPO7, YARS, TMPO, HNRNPH1, HLA-A, PDGFC, IL1B, S100A10, TXNDC2, DPY30, LAMP1, LYPLA1, CBR1, ARHGAP1, TPR, NAMPT, NAMPTL, CXCL2, EIF4A2, RPS4X, PABPN1, PHLDB1, NAP1L4, DDX6, PSMC3, MAP7D1, OSCAR, TES, NMT1, NMT2, MAT2A, PRMT1, COX17, SELM, CARS, PPP2CA, PPP2CB, VEGFC, VPS26A, PXN, PAWR, STRAP, RAB11A, RABI IB, EIF5, COPZ1, COPS3, CHMP4B, PDXK, RALA, RALB, SEC13, FHL2, TOMI, PYCARD, PDLIM4, RBBP4, RBBP7, RPS20, SRSF1, DLD, SNRPE, EIF4EBP1, RPLPO, RPLP0P6, LSM3, SLC16A3, CSE1L, NUCKS1, TSNAX, RPSA, RPSAP58, AP1B1, EIF2S1, LCP1,WSGR Ref. No.: 64320-709.601PSMD4, AARS, SRSF2, SFRS2, TRIPIO, CLTA, USO1, VBP1, UBE2K, MRPL12, UAP1, HIST1H1B, YBX3, RPL10A, IDH1, SNRPN, SNRPB, PCK2, EIF6, MMP3, DHX9, MATR3, UBE2D2, UBE2D3, PTRF, ACTR1A, LAMA1, PDCD6IP, QARS, HN1L, PLBD2, OS9, ILF3, ISOCI, PPP1R7, MAPK1, PDCD1LG2, GOLGB1, HSPA9, ACBD3, BTF3, UBE2I, SRRT, EIF4H, FERMT2, EIF3G, PARVA, DBNL, BOLA2, MAPRE1, PRDX5, STX12, EIF1, SMS, GRPEL1, OAF, SYNCRIP, ECE1, VAPA, PCNP, PSME1, EIF3J, C I4orf 166, DDX1, AKR1B1, SRP9, PCMT1, XPO1, MMP9, RPS7, NRCAM, FKBP3, SSB, DSC3, XRCC5, CCT6A, LIF, SERPINA9, SND1, ANP32B, G3BP1, GPC6, GLOD4, RRBP1, TGFB1, USP14, PLAUR, CXCL3, FASN, COPB2, TARDBP, TNFAIP6, RPS3, NDUFAB1, LMNB1, COL16A1, PSMF1, PHGDH, PRKCDBP, CXCL5, YAP1, TROVE2, MACF1, EIF5B, STX7, PFDNS, APOA1, PDAP1, STX12, EIF3K, FH, RBM8A, EIF1, RPS2, PTGR1, EEA1, CAB39, DCTN2, GDF15, MYL9, TCP1, RPS12, RAD23A, SKP1, PDLIM1, HSPH1, PSMB3, SUB1, API5, TWF2, KTN1, DYNC1H1, SNRNP70, MCFD2, PPP1R18, PTGES3, APEX1, TNKS1BP1, FUBP1, VAT1, PAFAH1B1, CBX1, PIK7, HNRPDL, ZNF185, DYNC1I2, KHDRBS1, SFPQ, PDCD5, TRIM28, PPIC, NARS, PCBP2, CD2AP, PSMA1, CAPRIN1, KHSRP, NSFL1C, PPP1R12A, NUDC, MFAP2, IGF2R, RGMB, PABPC1, MAN1A1, DDB1, PTBP1, FBN2, CNBP, OTUB1, NASP, HSPD1, EEF1D, RNPEP, CRKL, HNRNPAB, EIF3F, PAICS, ILF2, COPB1, PA2G4, COP A, NT5E, HMGA1, SF1, XRCC6, GNB2L1, MEI, MTAP, CCT2, ARCN1, PEPD, EPB41L3 PPP1CA, PPP1CC, STI 3. ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8-MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP, NAP1L1, HSP90AA1, SET, ENO1, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, 42254, and PAFAH1B2. In one instance, the secretome does not comprise 42254. Alternatively, or in addition, the method can optionally further comprise administering to the subject exosomes, wherein the exosomes are positive for protein expression of CD81 and / or CD63.

[0007] In some embodiments, the nMPCs are negative for:(i) a mutation in, or modified expression of, a SOD1 gene or RNA;(ii) a mutation in, or modified expression of, chromosome 9, open reading frame 72, optionally wherein the chromosome 9, open reading frame 72 comprises a C9ORF72 gene;(iii) a fused Fus gene mutation;(iv) a mutation in, or modified expression of, a TDP-43 gene or RNA;(v) a mutation in, or modified expression of, a STMN2 gene or RNA;(vi) a mutation in, or modified expression of, a UNC13A gene or RNA;(vii) a mutation in, or modified expression of, a TARDBP gene or RNA;(viii) a mutation in, or modified expression of, a hnRNPAl gene or RNA;(ix) a mutation in, or modified expression of, a hnRNPA2B 1 gene or RNA;(x) a mutation in, or modified expression of, a MATRS gene or RNA;WSGR Ref. No. : 64320-709.601(xi) a mutation in, or modified expression of, an ANG gene or RNA;(xii) a mutation in, or modified expression of, a TUBA4A gene or RNA;(xiii) a mutation in, or modified expression of, an ANXA11 gene or RNA;(xiv) a mutation in, or modified expression of, a PRPH gene or RNA;(xv) a mutation in, or modified expression of, a DCTN1 gene or RNA;(xvi) a mutation in, or modified expression of, a PFN1 gene or RNA;(xvii) a mutation in, or modified expression of, a KIF5a gene or RNA;(xviii) a mutation in, or modified expression of, a UBQLN2 gene or RNA;(xix) a mutation in, or modified expression of, a SQSTM1 gene or RNA;(xx) a mutation in, or modified expression of, an OPTN gene or RNA;(xxi) a mutation in, or modified expression of, a VCP gene or RNA;(xxii) a mutation in, or modified expression of, a CHMP2B gene or RNA;(xxiii) a mutation in, or modified expression of, a VAPBIVAMP gene or RNA; or(xxiv) a mutation in, or modified expression of, a TBK1 gene or RNA;(xxv) a mutation in, or modified expression of, an EphA4 gene or RNA;(xxvi) a mutation in, or modified expression of, a NEK-1 gene or RNA; or (xxvii) a mutation in, or modified expression of, an ATXN2 gene or RNA.

[0008] In one aspect, one or more symptoms of ALS are improved following treatment compared to treatment with a placebo or compared to the subject prior to administration of the nMPCs, nMPC secretome, nMPC exosomes or a combination thereof. In some embodiments, the one or more symptoms is improved (e.g., decreased) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to treatment with a placebo or compared to the subject prior to administration of the nMPCs, nMPC secretome, nMPC exosomes or a combination thereof. In other embodiments, the one or more symptoms is improved (e.g, decreased) by about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more compared to treatment with a placebo or compared to the subject prior to administration of the nMPCs, nMPC secretome, nMPC exosomes or a combination thereof.

[0009] In some embodiments, the nMPC secretome comprises a final protein concentration of from 0.001 mg / ml to about 0.1 mg / ml, 0. 1 mg / ml to about 50.0 mg / ml, or from about 0.5 mg / ml to about 20.0 mg / ml. In some embodiments, the nMPC exosomes comprise a final protein concentration of (a) at least about 0. 1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, about 2.0 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, about 11.0 mg / ml, about 12.0 mg / ml, about 13.0 mg / ml, about 14.0 mg / ml, about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 18.0 mg / ml, about 19.0 mg / ml, about 20.0 mg / ml, about 22.0 mg / ml, about 24.0 mg / ml, about 26.0 mg / ml, about 28.0 mg / ml, about 30.0 mg / ml, about 35.0 mg / ml, about 40.0 mg / ml, about 45.0 mg / ml, or about50.0 mg / ml, or (b) at most about 250.0 mg / ml, about 225.0 mg / ml, about 200.0 mg / ml, about 175 mg / ml, about 150 mg / ml, about 125 mg / ml, about 100 mg / ml, about 75 mg / ml, about 50 mg / ml, about 45.0 mg / ml, about 40.0 mg / ml, about 35.0 mg / ml, about 30.0 mg / ml,WSGR Ref. No. : 64320-709.601 about 25 mg / ml, about 20 mg / ml, about 15 mg / ml, about 10 mg / ml, about 5 mg / ml, about 1 mg / ml, about 0.5 mg / ml, about 0. 1 mg / ml, about 0.01 mg / ml, or about 0.001 mg / ml. In some embodiments, the nMPC secretome comprises a final protein concentration of at least about 0.01 mg / ml±0.05. In some embodiments, the secretome comprises comprise a final protein concentration of at least about 0.001 mg / ml±0.005. In some embodiments, the secretome comprises comprise a final protein concentration of at least about 1.0 mg / ml±0.5. In some embodiments, the nMPC secretome comprises a final protein concentration of at least about 1.0 mg / ml±0.5. In other embodiments, the nMPC secretome comprises a final protein concentration of at least about 4.0 mg / ml±1.0. In some embodiments, the nMPC secretome comprises a final protein concentration of at least about 8.0 mg / ml±2.0. In yet other embodiments, the nMPC secretome comprises a final protein concentration of at least about 16.0 mg / ml±3.0. In some instances, the composition is concentrated. In some instances, the composition is diluted prior to the administration. In some instances, the nMPC secretome is administered at a concentration of at least about 10 pg / ml to at least about 1000 pg / ml.

[0010] In some embodiments, a symptom of ALS is improved following treatment with the secretome compared to treatment with a placebo or compared to the subject prior to administration of the secretome. In some embodiments, the one or more symptoms is improved (e.g, decreased) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to treatment with a placebo or compared to the subject prior to administration of the secretome. In other embodiments, the one or more symptoms is improved (e.g., decreased) by about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, or more compared to treatment with a placebo or compared to the subject prior to administration of the secretome.

[0011] In some embodiments, the nMPC exosomes comprise particles in a range of from about 50 nm to about 1000 nm, about 100 nmto about 1000 nm, about 500 nmto about 1000 nm, about 600 nm to about 900 nm, about 700 nm to about 800 nm, about 300 nm to about 600 nm, about 300 nm to about 500 nm, 300 nm to about 400 nm, about 200 nm to about 500 nm, 200 nm to about 400 nm, about 100 nm to about 500 nm, about 100 nm to about 300 nm, 100 nm to about 200 nm, 100 nm to about 150 nm, about 50 nm to about 200 nm, about 50 nm to about 150 nm, about 50 nm to about 110 nm, about 50 nm to about 100 nm, about 75 nm to about 250 nm, about 75 nm to about 200 nm, about 75 nm to about 150 nm, about 75 nm to about lOO nm, about 90 nm to about 150 nm, about 90 nmto about 125 nm, about 90 nm to about 110 nm, or about 90 nm to about 100 nm in diameter. In one embodiment, the nMPC exosomes comprise particles in a range of from about 90 nm to about 110 nm in diameter. In another embodiment, the nMPC exosomes comprise particles in a range of from about 100 nm to about 120 nm in diameter. In some embodiments, the nMPC exosomes comprise particles about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, lOO nm, HO nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm in diameter. In some embodiments, the nMPC exosomes comprise particles about 90 nm in diameter. In certain embodiments, the nMPC exosomes comprise particles about 100 nm in diameter. In other embodiments, the nMPC exosomes comprise particles aboutWSGR Ref. No. : 64320-709.601110 nm in diameter. In yet other embodiments, the nMPC exosomes have a density of about 1.13 g / ml to about 1. 19 g / ml.

[0012] In some embodiments, the nMPC exosomes comprise a final protein concentration of from about 0.001 mg / ml to about 0.1 mg / ml, 0. 1 mg / ml to about 50.0 mg / ml, or from about 0.5 mg / ml to about 20.0 mg / ml. In some embodiments, the nMPC exosomes comprise a final protein concentration of (a) at least about 0.001 mg / ml, 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, about 2.0 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, about 11.0 mg / ml, about 12.0 mg / ml, about 13.0 mg / ml, about 14.0 mg / ml, about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 18.0 mg / ml, about 19.0 mg / ml, about 20.0 mg / ml, about 22.0 mg / ml, about 24.0 mg / ml, about 26.0 mg / ml, about 28.0 mg / ml, about 30.0 mg / ml, about 35.0 mg / ml, about 40.0 mg / ml, about 45.0 mg / ml, or about 50.0 mg / ml, or (b) at most about 250.0 mg / ml, about 225.0 mg / ml, about 200.0 mg / ml, about 175 mg / ml, about 150 mg / ml, about 125 mg / ml, about 100 mg / ml, about 75 mg / ml, about 50 mg / ml, about 45.0 mg / ml, about 40.0 mg / ml, about 35.0 mg / ml, about 30.0 mg / ml, about 25 mg / ml, about 20 mg / ml, about 15 mg / ml, about 10 mg / ml, about 5 mg / ml, about 1 mg / ml, about 0.5 mg / ml, about 0.1 mg / ml, about 0.01 mg / ml, or about 0.001 mg / ml. In some embodiments, the nMPC exosomes comprise a final protein concentration of at least about 0.01 mg / ml±0.05. In some embodiments, the nMPC exosomes comprise a final protein concentration of at least about 0.001 mg / ml±0.005. In some embodiments, the nMPC exosomes comprise a final protein concentration of at least about 1.0 mg / ml±0.5. In other embodiments, the nMPC exosomes comprise a final protein concentration of at least about 4.0 mg / ml±1.0. In some embodiments, the nMPC exosomes comprise a final protein concentration of at least about 8.0 mg / ml±2.0. In certain embodiments, the nMPC exosomes comprise a final protein concentration of at least about 16.0 mg / ml±3.0. In some instances, the composition is concentrated. In some instances, the composition is diluted prior to the administration. In some instances, the nMPC exosomes are administered at a concentration of at least about 10 pg / ml to at least about 1000 pg / ml. Alternatively, or in addition, in certain embodiments, the nMPC secretome and / or the nMPC exosomes are positive for protein expression of one or more of the proteins in Table 4.

[0013] In one aspect, the composition comprises an nMPC total secretome comprising the nMPC secretome and the nMPC exosomes. In another aspect, the subject is administered a composition comprising the nMPC total secretome comprising the nMPC secretome and the nMPC exosomes. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of from about 0. 10 mg / ml to about 50.0 mg / ml, or from about 0.50 mg / ml to about 20.0 mg / ml. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of (a) at least about 0.001 mg / ml, 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, about 2.0 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, about 11.0 mg / ml, about 12.0 mg / ml, about 13.0 mg / ml, about 14.0 mg / ml, about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 18.0 mg / ml, about 19.0 mg / ml, about 20.0 mg / ml, about 22.0 mg / ml, about 24.0 mg / ml, about 26.0 mg / ml,WSGR Ref. No. : 64320-709.601 about 28.0 mg / ml, about 30.0 mg / ml, about 35.0 mg / ml, about 40.0 mg / ml, about 45.0 mg / ml, or about 50.0 mg / ml, or (b) at most about 250.0 mg / ml, about 225.0 mg / ml, about 200.0 mg / ml, about 175 mg / ml, about 150 mg / ml, about 125 mg / ml, about 100 mg / ml, about 75 mg / ml, about 50 mg / ml, about 45.0 mg / ml, about 40.0 mg / ml, about 35.0 mg / ml, about 30.0 mg / ml, about 25 mg / ml, about 20 mg / ml, about 15 mg / ml, about 10 mg / ml, about 5 mg / ml, about 1 mg / ml, about 0.5 mg / ml, about 0. 1 mg / ml, about 0.01 mg / ml, or about 0.001 mg / ml. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of at least about 0.01 mg / ml±0.05. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of at least about 0.001 mg / ml±0.005. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of at least about 1.0 mg / ml±0.5. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of at least about 4.0 mg / ml±1.0. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of at least about 8.0 mg / ml±2.0. In some embodiments, the nMPC secretome and the nMPC exosomes collectively comprise a final protein concentration of at least about 16.0 mg / ml±3.0. In some instances, the nMPC secretome and the nMPC exosomes collectively are administered at a concentration of at least about 10 pg / ml to at least about 1000 pg / ml. Alternatively, or in addition, in certain embodiments, the nMPC secretome and / or the nMPC exosomes are positive for protein expression of one or more of the proteins in Table 4. In some embodiments, the composition is acellular.

[0014] Symptoms of ALS to be treated include, but are not limited to, shortness of breath (e.g, difficulty breathing), fatigue, muscle weakness (e.g, feeling tired, even after resting), muscle twitching (e.g., twitching, cramping, or spasms in the muscles of the hands, feet, arms, shoulders, tongue, etc.), speech difficulties (e.g., slurred, nasal, or thick speech, difficulty projecting the voice, etc.), difficulty swallowing or chewing, trouble walking (e.g, tripping, falling, etc.), uncontrollable periods of laughing or crying, and / or loss of motor control (e.g., drooling, difficulty raising the foot, clumsiness, dropping things, paralysis, constipation, etc.). Treatment may, in some instances, increase the subject’s quality of life compared to treatment with a placebo or compared to the subject prior to administration of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof. Alternatively, or in addition, treatment may increase the subject’s lifespan compared to treatment with a placebo or compared to the subject prior to administration of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof. In certain embodiments, treatment improves one or more of: overall inflammation, chronic inflammation, inflammation in the nervous system, high levels of pro- inflammatory factors, oxidative stress, abnormal protein clumping, abnormal protein production, excess glutamate production, pro-inflammatory actions of microglia and astrocytes, motor neuron condition, muscle twitching or cramping, motor control in the hands and arms, fatigue, speech, breathing, swallowing etc. In certain instances, one symptom is improved following treatment. In certain instances, two symptoms are improved following treatment. In certain instances, three (3) symptoms are improved following treatment. In certain instances, four (4) symptoms are improved following treatment. In certainWSGR Ref. No. : 64320-709.601 instances, five (5) symptoms are improved following treatment. In certain instances, six (6) or more symptoms are improved following treatment. Treatment may, in some instances, increase the subject’s quality of life compared to treatment with a placebo or compared to the subject prior to treatment. Alternatively, or in addition, treatment may increase the subject’s lifespan (e.g., quantity) compared to treatment with a placebo or compared to the subject prior to treatment. In certain embodiments, treatment results in stasis of one or more symptoms (e.g, improving less than 2% or not getting worse). In some embodiments, the subject exhibits an improvement in shortness of breath (e.g, difficulty breathing), fatigue, muscle weakness (e.g, feeling tired, even after resting), muscle twitching (e.g., twitching, cramping, or spasms in the muscles of the hands, feet, arms, shoulders, tongue, etc.), speech difficulty (e.g., slurred, nasal, or thick speech, difficulty projecting the voice, etc.), difficulty swallowing, difficulty chewing, trouble walking (e.g., tripping, falling, etc.), uncontrollable periods of laughing, uncontrollable periods of crying, uncontrollable periods of loss of motor control (e.g, drooling, difficulty raising the foot, clumsiness, dropping things, paralysis, constipation, etc.), or a combination thereof, or a combination thereof following administration of allogeneic, human, neonatal cardiac mesenchymal progenitor cells (nMPCs), nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof. In certain embodiments, the symptom further comprises neurodegeneration, inflammation, oxidative stress, or a combination thereof. In one nonlimiting example, treatment reduces neurodegeneration. In one instance, treatment reduces inflammation by at least 2% or by at least 2-fold as compared to treatment with a placebo or compared to the subject prior to treatment. In another instance, treatment reduces oxidative stress by at least 2% or by at least 2-fold as compared to treatment with a placebo or compared to the subject prior to treatment.

[0015] In some embodiments, administration of any composition comprising nMPCs, nMPC secretome, nMPC exosomes or a combination thereof as described herein comprises intravenous (IV) injection, intracranial injection, intracerebroventricular (ICV) injection, intrathecal (IT) injection, intramuscular (IM) injection, or intravenous (IV) catheter (e.g, intravenous (IV) infusion). In some embodiments, nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof are administered into the brain of the subject via intracranial transplantation. In some embodiments, the method comprises treating a subject diagnosed with Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the method comprises treating a subject diagnosed with Amyotrophic Lateral Sclerosis (ALS) with nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof as described herein. In some embodiments, the method comprises treating a subject diagnosed with ALS with a secretome described herein. In some embodiments, the method comprises treating a subject diagnosed with ALS by intravenous delivery of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof as described herein.

[0016] In some embodiments, the nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof are administered to the subject once every twenty-eight (28) days. In some embodiments, the nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof are administered to the subject once every 29, 30, 31, 32, 33, 34, 35, or 36 days. In someWSGR Ref. No. : 64320-709.601 embodiments, the nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof are administered to the subject for from about 1 month to about 10 years, or from about 1 year to about 50 years. In one embodiment, a dose of the nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof is administered to the subject about every twenty-eight (28) days or about every thirty (30) days. In some embodiments, the subject is administered a first dose of the nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof at day 0 and a second dose of the nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof at day 28. In some embodiments, the subject is assessed at day 7 following treatment.

[0017] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent or therapy. In some embodiments, the additional therapeutic agent helps manage some symptoms of ALS. In some embodiments, the method further comprises administering the nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof concomitantly with other standard of care treatment. In some embodiments, the method further comprises administering the nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof concomitantly with other standard of care treatment (e.g., physical, occupational, speech, respiratory, and nutritional therapies, mechanical ventilation, etc. ).

[0018] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In certain embodiments, the subject to be treated is a child of from birth to 18 years of age. In some embodiments, the subject to be treated is an adult of 18 years of age or older. In some embodiments, the subject to be treated is a child from about 1 to about 4 years of age, or from about 5 to about 14 years of age. In some embodiments, the subject to be treated is from about 15 to about 40 years of age, or 41 years of age or older. In certain embodiments, the subj ect to be treated is from about 4 to about 18 years of age. In certain embodiments, the subject to be treated is from about 19 to about 25 years of age. In certain embodiments, the subject to be treated is 26 years of age or older. In some embodiments, the subject is from about 1 to about 10 years of age, from about 11 to about 20 years of age, from about 21 to about 30 years of age, from about 31 to about 40 years of age, from about 51 to about 60 years of age, from about 71 to about 80 years of age, from about 81 to about 90 years of age, or about 91+ years of age.

[0019] In any of such methods, the nMPCs are allogeneic. In any of such methods, the subject to be treated can be a mammal such as, for example, a human. In some embodiments, the nMPCs are infused via IV catheter for about 60 minutes to about 180 minutes. In some embodiments, the subject is screened for a symptom at one or more times prior to treatment, during treatment, and / or after treatment. In some embodiments, the screening comprises a physical evaluation, medical history review, or a diagnostic test. In certain embodiments, the diagnostic test comprises an electromyography (EMG), a nerve conduction study, a blood test, a urine test, or an MRI scan.

[0020] In some embodiments, one or more proinflammatory cytokines in the subject are reduced by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In certain embodiments, the one or more proinflammatory cytokines comprise IL-1 , IL-6, IL-8, IL-12, IL-17a, IL-WSGR Ref. No.: 64320-709.60118, MCP-1, TNF-a, IFN-y, or a combination thereof. In one embodiment, the one or more proinflammatory cytokines comprise IL-1 , IL-6, IL-8, IL-12, IL-17a, IL-18, MCP-1, TNF-a, and IFN-y. In another embodiment, the one or more proinflammatory cytokines comprise IL-8, IL-17a, MCP-1, TNF-a, and IFN-y. In yet another embodiment, the one or more proinflammatory cytokines comprise IL- 8, MCP-1, and TNF-a. In one embodiment, the one or more proinflammatory cytokines comprise IL-17a and IFN-y. In some embodiments, the administration of the allogeneic, human, neonatal cardiac mesenchymal progenitor cells (nMPCs), nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof, inhibits IL- 10, IL-6, IL-8, IL- 12, IL- 17a, IL- 18, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-a, interferon gamma (IFN-y), or a combination thereof by at least 2% or by at least 2-fold as compared to treatment with a placebo or compared to the subject prior to treatment.

[0021] In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is above about 10 pg / ml, about 20 pg ml, about 30 pg / ml, about 40 pg / ml, or about 50 pg / ml prior to treatment. In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is reduced by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream of the subject is reduced by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the cerebrospinal fluid (CSF) of the subject is reduced by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is reduced by at least about 1 pg / ml, 2 pg / ml, 3 pg / ml, 4 pg / ml, 5 pg / ml, 6 pg / ml, 7 pg / ml, 8 pg / ml, 9 pg / ml, 10 pg / ml, about 20 pg ml, about 30 pg / ml, about 40 pg / ml, or about 50 pg / ml compared to treatment with a placebo or compared to the subject prior to treatment.

[0022] In some embodiments, a level of interleukin 8 (IL-8), lipopolysaccharide binding protein (LBP), cluster of differentiation 14 (CD14), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), Insulin-like Growth Factor 1 (IGF-1), Epidermal Growth Factor (EGF), Transforming Growth Factor Beta 1 (TGF-01), TGF-02, TGF-03, neurotrophin-3 (NTF-3), hepatocyte growth factor (HGF), growth differentiation factor 15 (GDF-15), heparin-binding epidermal growth factor-like growth factor (HB-EGF), chitinase-3-like protein 1 (CHI3L1) (Gp39), connexin 39 (Cx39), Cyclophilin B (CypB) (PPIB), TATA-Binding Protein (TBP), DNA-directed RNA polymerase II subunit RPB1 (POLR2A), Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), beta actin (ACTB), or a combination thereof of the subject is modulated by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of IL-8, LBP, CD14, BDNF, GDNF, IGF-1, TGF-01, TGF-02, TGF-03, GDF-15, HB-EGF, CHI3L1 (Gp39), Cx39, PPIB, ACTB, or a combination thereof of the subject is reduced by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of EGF, PPIB, GAPDH, or aWSGR Ref. No. : 64320-709.601 combination thereof of the subject is increased by at least 2% compared to treatment with a placebo or compared to the subj ect prior to treatment. In some embodiments, a level of interleukin 1 f> (IL- 1 P) , IL-6, IL- 10, TNF-a, or a combination thereof of the subject is modulated by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment.

[0023] In some embodiments, a level of Phosphorylated Neurofilament Heavy Chain (pNfH), TDP-43, FUS, SOD1, a dipeptide repeat (DPR) protein, tau protein, phosphorylated tau protein (p-tau), clusterin, a chitinase-like protein, creatine kinase (CK), Glial Fibrillary Acidic Protein (GFAP), or a combination thereof of the subject is modulated by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Phosphorylated Neurofilament Heavy Chain (pNfH), TDP-43, FUS, SOD1, a dipeptide repeat (DPR) protein, tau protein, phosphorylated tau protein (p-tau), clusterin, a chitinase-like protein, creatine kinase (CK), Glial Fibrillary Acidic Protein (GFAP), or a combination thereof of the subject is reduced by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment.

[0024] In some embodiments, the subject has an improved quality of life or an improved length of life following treatment compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, the subject is biologically a male. In some embodiments, the subject is biologically a female.

[0025] In any of such methods, the nMPCs are stable in storage at a temperature for a period of time. In some embodiments, the temperature is about 4 °C, 1 °C, 0 °C, -10 °C, -20 °C, or -80 °C. In some embodiments, the period of time is at least about 1, 2, 3, 4, 5, 6, or 7 days; at least about 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; or at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months. In some embodiments, the nMPCs are stored in cell cryopreservation media- CS10 (CRYOSTOR®; Sigma- Aldrich, Israel). In some embodiments, the nMPCs are thawed in a 37 °C water bath until a single ice crystal remained prior to administration to the subject. In some embodiment^ the nMPCs are further washed with a volume of Complete Medium (CM). In some embodiments, the nMPCs are washed twice with a volume of Complete Medium (CM). In some embodiments, the volume is at least about 10 ml.

[0026] In some embodiments, the cry opreservation medium comprises a permeating cryoprotective agent. In some embodiments, the permeating cryoprotective agent comprises a US Pharmacopeia (USP)- grade permeating cryoprotective agent. In some embodiments, the cryopreservation medium comprises about 10% of the permeating cryoprotective agent. In some embodiments, the cry opreservation medium comprises about 10% of a Dimethyl Sulfoxide (DMSO). In some embodiments, the cryopreservation medium comprises a US Pharmacopeia (USP)-grade DMSO. In some embodiments, the cry opreservation medium is serum-free, protein-free, sterile, free of toxins, GMP certified, free of endotoxins, or free of pathogens.

[0027] In some embodiments, a unit dose comprises a composition of the nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof as described herein. In someWSGR Ref. No. : 64320-709.601 embodiments, an intravenous (IV) bag comprises a composition of the nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof as described herein. In some embodiments, the IV bag comprises a total of from about 50 million to about 250 million cells. In some embodiments, a human subject is administered with one or more IV bags comprising nMPCs. In some embodiments, when more than one IV bag is administered, each bag is administered at about every 28 days.

[0028] In some embodiments, the subject is administered the nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof at a dosage of at least about 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 22.5 mg / kg, 25 mg / kg, 27.5 mg / kg, 30 mg / kg, 32.5 mg / kg, 35 mg / kg, 37.5 mg / kg, 40 mg / kg, 42.5 mg / kg, 45 mg / kg, 47.5 mg / kg, or 50 mg / kg, where mg / kg is the nMPC secretome or nMPC exosome protein weight per kilogram of subject body weight.

[0029] Provided herein, in some aspects, is a composition comprising allogeneic, human nMPCs, wherein the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM- 1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. In some embodiments, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2. In certain embodiments, the nMPCs are further negative for protein expression of pl6INK4aup to at least passage 8.

[0030] In some embodiments, a composition as described herein further comprises an nMPC secretome (conditioned medium) or nMPC exosomes. In certain embodiments, the composition further comprises an nMPC secretome (conditioned medium) and nMPC exosomes. In some embodiments, the nMPC secretome (conditioned medium) or nMPC exosomes are positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and / or Akt. In some embodiments, the nMPC secretome comprises HSF1 at levels higher than HSF1 in the secretome of aMPCs. In some embodiments, the secretomes or exosomes are negative for protein expression of IGF 1. In some embodiments, the nMPCs are genetically engineered. In some embodiments, the nMPCs are genetically engineered to overexpress one or more paracrine factors. In some embodiments, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1. In one embodiment, the nMPCs are genetically engineered to overexpress 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1. In another embodiment, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1.

[0031] In some embodiments, the nMPCs are non-naturally occurring. In some embodiments, the nMPCs are allogeneic. In some embodiments, the nMPCs are not xenogenic. In some embodiments, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, andNkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4. In some embodiments, the nMPC secretomes or nMPC exosomesWSGR Ref. No. : 64320-709.601 are positive for protein expression of one or more of the proteins in Table 4. In some embodiments, the cells are obtained from a subject who is biologically a female. In certain embodiments, the cells are obtained from a subject who is biologically a male. In some embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, or 6 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5; (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4; (c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / or (d) negative for protein expression of p^giNK4aUpl0 al|easlpassage g |nsome embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs.

[0032] Provided herein, in some aspects, are cryopreserved, allogeneic, human nMPCs, wherein the cryopreserved, allogeneic, human nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4. In some embodiments, the cryopreserved, allogeneic, human nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2. In certain embodiments, the cryopreserved, allogeneic, human nMPCs are further negative for protein expression of pl6INK4aup to at least passage 8. In one embodiment, the composition further comprises cryopreserved secretomes or exosomes. In another embodiment, the composition further comprises cryopreserved secretomes and cryopreserved exosomes. In one embodiment, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, or 8 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, andNkx2.5; (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4; (c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / or (d) negative for protein expression of pl6INK4aup to at least passage 8. In another embodiment, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), CD44, CD73, CD47, Ki67, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. In some embodiments, the nMPCs are negative for: (i) a mutation in, or modified expression of, a SOD! gene or RNA; (ii) a mutation in, or modified expression of, chromosome 9, open reading frame 72, optionally wherein the chromosome 9, open reading frame 72 comprises a C9ORF72 gene; (iii) a fused Fus gene mutation; (iv) a mutation in, or modified expression of, a TDP-43 gene or RNA; (v) a mutation in, or modified expression of, a STMN2 gene or RNA; (vi) a mutation in, or modified expression of, a UNC13A gene or RNA; (vii) a mutation in, or modified expression of, a TARDBP gene or RNA; (viii) a mutation in, or modified expression of, ahnRNPAl gene or RNA; (ix) a mutation in, or modified expression of, a hnRNPA2Bl gene or RNA; (x) a mutation in, orWSGR Ref. No. : 64320-709.601 modified expression of, aMATR3 gene or RNA; (xi) a mutation in, or modified expression of, an ANG gene or RNA; (xii) a mutation in, or modified expression of, a TUBA4A gene or RNA; (xiii) a mutation in, or modified expression of, an ANXA11 gene or RNA; (xiv) a mutation in, or modified expression of, a PRPH gene or RNA; (xv) a mutation in, or modified expression of, a DCTN1 gene or RNA; (xvi) a mutation in, or modified expression of, a PFN1 gene or RNA; (xvii) a mutation in, or modified expression of, aKIF5a gene or RNA; (xviii) a mutation in, or modified expression of, a UBQLN2 gene or RNA; (xix) a mutation in, or modified expression of, a SQSTM1 gene or RNA; (xx) a mutation in, or modified expression of, an OPTN gene or RNA; (xxi) a mutation in, or modified expression of, a VCP gene or RNA; (xxii) a mutation in, or modified expression of, a CHMP2B gene or RNA; (xxiii) a mutation in, or modified expression of, a VAPBiVAMP gene or RNA; or (xxiv) a mutation in, or modified expression of, a TBK1 gene or RNA; (xxv) a mutation in, or modified expression of, an EphA4 gene or RNA; (xxvi) a mutation in, or modified expression of, aNEK-1 gene or RNA; or (xxvii) a mutation in, or modified expression of, an ATXN2 gene or RNA.INCORPORATION BY REFERENCE

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE FIGURES

[0034] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0035] FIGS. 1A-1E are in vitro data assessing the effect of nMPC secretomes on LPS -activated microglia and their expression levels of exemplary inflammatory markers. FIG. 1A is a histogram showing the concentration of TNF-a (pg / ml) in controls and after treatment of LPS-stimulated microglia with 1 or 10 pg / ml nMPC secretome. FIG. IB is a histogram showing the concentration of IL-8 (pg / ml) in controls and after treatment of LPS-stimulated microglia with 1 or 10 pg / ml nMPC secretome. FIG. 1C is a histogram showing the concentration of MCP-1 (pg / ml) in controls and after treatment of LPS- stimulated microglia with 1 or 10 pg / ml nMPC secretome. FIG. ID is a histogram showing the concentration of IL- 17a (pg / ml) in controls and after treatment of LPS -stimulated microglia with 1 or 10 pg / ml nMPC secretome. FIG. IE is a histogram showing the concentration of IFN-y (pg / ml) in controls and after treatment of LPS-stimulated microglia with 1 or 10 pg / ml nMPC secretome. Left panels in each histogram represent data collected 24 hours after treatment with nMPC secretome, and right panels represent data collected 48 hours after treatment with nMPC secretome. Bars marked with circlesWSGR Ref. No. : 64320-709.601 represent data for controls. Bars marked with triangles represent microglia stimulated with LPS (positive control). Bars marked with diamonds represent LPS -stimulated microglia treated with 1 pg / ml nMPC secretome. Bars marked with pentagons represent LPS-stimulated microglia treated with 10 pg / ml nMPC secretome.

[0036] FIG. 2 is a schematic of the survival study arm paradigm.

[0037] FIG. 3 is a schematic of the sampling study arm paradigm.

[0038] FIG. 4 are exemplary lumbar spinal cord endpoint collection details for immunohistochemical analysis.

[0039] FIG. 5 is a photograph of an exemplary spinal cord orientation in a tissue cassette with pre-cut foam. The segment is marked using a tissue ink at the caudal end and caudal (inked) end is positioned towards the cassette label.

[0040] FIG. 6 is a schematic of an embedding plan for the spinal cords. n=3 lumbar spinal cord segments from three separate animals will are embedded into a single paraffin block.DETAILED DESCRIPTION

[0041] The details of one or more inventive embodiments are set forth in the accompanying drawings, the claims, and the description herein. Other features, objects, and advantages of the inventive embodiments disclosed and contemplated herein can be combined with any other embodiment unless explicitly excluded. It is to be understood that the embodiments of the disclosures herein are illustrative of the principles of the present disclosure. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto. Any combination of the abovedescribed elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure may be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

[0042] The present disclosure provides, among other things, methods and compositions for treating Amyotrophic Lateral Sclerosis (ALS), based on neonatal cardiac mesenchymal progenitor cells (nMPCs) nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof as therapeutics. In particular embodiments, nMPC secretomes (conditioned medium) can comprise exosomes, growth factors, cytokines, macromolecules, such as peptides, proteins, and nucleic acids (e.g, miRNAs), or a combination thereof. In some embodiments, the present disclosure provides methods of treating a symptom of cardiomyopathy in ALS, including, for example, administering to an individual who is suffering from or susceptible to ALS an effective amount of nMPCs, nMPC secretome, nMPC exosomes or a combination thereof such that at least one symptom or feature of ALS is reduced by at least 2% or by at least 2-fold in intensity, severity, or frequency, or has delayed onset compared to treatment with a placebo.WSGR Ref. No. : 64320-709.601Amyotrophic Lateral Sclerosis

[0043] Amyotrophic Lateral Sclerosis (ALS) is arare disease with a prevalence of 4-6 for every 100,000 people each year and an incidence of 1-2 for every 100,000 people each year. About 90% of ALS cases are classified as sporadic ALS (SALS), and the remaining 10% are inherited and referred to as familial ALS (FALS), with a Mendelian pattern of inheritance. Familial (FALS) and sporadic (SALS) cases are clinically difficult to distinguish from one another. The causes for most cases of ALS are unknown and the clinical progression is highly variable, suggesting that multiple factors underlie the disease mechanism. Few treatments are available.

[0044] Characteristic of this disease is the selective death of motor neurons located in the brainstem, motor cortex and spinal cord leading to paralysis of voluntary muscles. The paralysis begins focally and disseminates in a pattern that suggests that degeneration spreads among contiguous pools of motor neurons. Mortality normally results when control of the diaphragm is impaired and the ability to breathe is lost.

[0045] ALS is characterized by progressive manifestations of dysfunction of both lower and upper motor neurons. Lower motor neurons connect the brainstem and spinal cord to muscle fibers. Their dysfunction leads to muscle atrophy, cramps, and fasciculations (small, local, involuntary muscle contraction). Upper motor neurons originate in motor region of the cerebral cortex or the brainstem and carry motor information down to motor neurons that are responsible for stimulating the target muscle. Their dysfunction leads to spasticity (continuous muscle contraction that interfere with gait, movement, and speech, etc.) and pathological reflexes. The other related motor neuron diseases are usually distinguished by the type of nerve cells impaired, i. e., upper or lower motor neurons.

[0046] Disclosed herein are compositions and formulations of neonatal cardiac mesenchymal progenitor cells (nMPCs), nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof that can reverse or inhibit (completely or partially) pathophysiological hallmarks and symptoms of Amyotrophic Lateral Sclerosis (ALS) (e.g, inflammation or oxidative stress in the neurons or muscles). Alternatively, or in addition, disclosed herein are compositions and formulations of nMPC secretomes which reverse or inhibit (completely or partially) pathophysiological hallmarks and symptoms of ALS. Alternatively, or in addition, disclosed herein are compositions and formulations of nMPC exosomes which reverse or inhibit (completely or partially) pathophysiological hallmarks and symptoms of ALS. Different cell types mediating neuroinflammatory processes, such as microglia and astrocytes, are thought to be involved in the progression of the disease. Stress and injuries appear to aggravate pro- inflammatory microglial activity, and inhibition of microglial function or interfering with microglial reactivity may help slow the progression of neuronal degeneration in the disease progression of ALS.

[0047] In one aspect, described herein are methods which inhibit (e.g, reduce) one or more proinflammatory cytokines including, but not limited to, Interleukin (IL)-1|3, IL-6, IL-8, IL-12, IL-17a, IL-18, MCP-1, tumor necrosis factor (TNF)-a, interferon gamma (IFN-y). In one embodiment, the downregulated cytokines comprise TNF-a, IL-8, MCP-1, IL-17a, and IFN-y. In another embodiment, theWSGR Ref. No. : 64320-709.601 downregulated cytokines comprise TNF-a, IL-8, and MCP-1. In yet another embodiment, the downregulated cytokines comprise IL-17a and IFN-y.

[0048] Progenitor cell-derived therapies with the cells described herein, secretomes, and / or exosomes described herein, which have a multimodal mechanism of action capable of targeting the critical pathological mechanisms of ALS, represent a promising therapeutic approach for reducing the inflammatory processes or oxidative stress associated with this disease. nMPCs, nMPC secretome, nMPC exosomes or a combination thereof can help reduce levels of inflammation, reduce levels of pro- inflammatory factors, and / or reduce levels of oxidative stress. The compositions comprising nMPCs, nMPC secretome, nMPC exosomes or a combination thereof can offset the pathophysiological consequences of neurodegeneration by recruiting regenerative factors and / or targeting inflammation. nMPCs, nMPC secretome, nMPC exosomes or a combination thereof described herein can reduce levels of inflammation, reduce levels of pro-inflammatory factors, and / or reduce levels of oxidative stress. In some embodiments, reduction is at least 2% or more compared to a placebo or compared to a subject prior to treatment. In other embodiments, reduction is at least 2-fold or more compared to a placebo or compared to a subject prior to treatment. The goal of using the compositions comprising nMPCs, nMPC secretome, nMPC exosomes or a combination thereof described herein is to offset pathophysiological consequences of degeneration of a subject by partially or treating a symptom of ALS.

[0049] nMPCs, nMPC secretome, nMPC exosomes or a combination thereof disclosed herein comprise an allogeneic, culture-expanded neonatal cardiac mesenchymal progenitor cell (nMPC) formulation with potent immunomodulatory effects and the potential to block cytokine storms. These nMPCs are isolated from the right atrial appendage of neonatal (<30 days old) donors with normal myocardium undergoing congenital heart surgery. In certain instances, therapeutic benefits of nMPCs can be exerted through their secretome, which is comprised of, for example, independently secreted growth factors, cytokines, chemokines, and / or miRNA-enriched exosomes. Robust analyses, including deep proteomic analysis and RNA sequencing of the nMPC secretome coupled with Ingenuity Pathway Analysis, have shown the presence of unique proteins and miRNA within the secretome may be responsible for downregulating fibrotic, inflammatory, and oxidative stress pathways (ERK / MAPK Signaling pathway) while promoting cellular growth, survival, and proliferation pathways. Thus, nMPCs, nMPC secretome, nMPC exosomes or a combination thereof operate through a multimodal mechanism of action, enhancing pathways related to angiogenesis, immunomodulation, energy production, and cardiomyocyte proliferation while reducing oxidative stress, inflammation, and / or fibrosis.

[0050] nMPCs, nMPC secretome, nMPC exosomes or a combination thereof disclosed herein have demonstrated superior therapeutic potential compared to other well -characterized and clinically relevant stem / progenitor cell types in vitro and in vivo. The nMPCs significantly inhibited secretion of pro- inflammatory cytokines TNF-a, IL-8, and MCP-1. IL- 17a and IFN-y were also shown to be reduced following exposure to nMPCs.

[0051] Neuronal degeneration, inflammation, and oxidative stress lead to clinical features such as muscle weakness, respiratory difficulties, and / or increased risk of cardiac death in ALS. Thus, theWSGR Ref. No. : 64320-709.601 potential anti-inflammatory effects of nMPCs can provide relevant insights into the potential benefit of nMPCs in ALS and support evaluating the impact of nMPCs on this disease.

[0052] In various embodiments, a damaged or dysfunctional tissue (e.g, neuronal tissue) may need repair, regeneration, or improved function due to chronic inflammation or degeneration. In some embodiments, administration of a composition as described herein comprising nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof can result in a decrease in inflammatory factors or factors associated with oxidative stress.

[0053] Provided herein is a method of treating ALS in a subject in need thereof, comprising administering neonatal cardiac mesenchymal progenitor cells (nMPCs) to the subject, wherein the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM- 1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4. In one non-limiting embodiment, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. In one embodiment, the nMPCs are non-naturally occurring. In some embodiments, the subject is nonambulatory. In other embodiments, the subject is ambulatory. In some embodiments, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2. In some embodiments, the nMPCs are further negative for protein expression of p!6INK4aup to at least passage 8. In some embodiments, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF - 1 a, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1. In some embodiments, the nMPCs are genetically engineered. In some embodiments, the nMPCs are genetically engineered to overexpress one or more paracrine factors. In some embodiments, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. In some embodiments, the nMPCs are non-naturally occurring. In some embodiments, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4. In one aspect, the subject treated by the method exhibits an improvement of one or more symptoms of cardiomyopathy by about 2% or more compared to treatment with a placebo or compared to the subject prior to administration of the nMPCs, nMPC secretome, nMPC exosomes or a combination thereof). In any of such methods, the nMPCs are allogeneic. In any of such methods, the subject to be treated can be a mammal such as, for example, a human. The human can be a child (from birth to 18 years of age) or an adult (over 18 years of age).Neonatal Cardiac Mesenchymal Progenitor Cells (nMPCs)

[0054] In one aspect, neonatal cardiac mesenchymal progenitor cells (nMPCs) for use in the methods described herein are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM- 1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4. In another embodiment, nMPCs are further negativeWSGR Ref. No. : 64320-709.601 for protein expression of p!6INK4aup to at least passage 8. In one instance, nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4. Alternatively, or in addition, nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2. In one non-limiting embodiment, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, or SOX2. In another non-limiting embodiment, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2. Alternatively, or in addition, nMPCs can be further negative for protein expression of p!6iNK4aUpl0 al|easlpassageg |n oneembodiment, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and / or pl6INK4a. In one instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, or SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, or pl6INK4a. In another instance, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and p!6INK4a. In some embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, or 8 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5; (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4; (c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / or (d) negative for protein expression of p!6INK4aup to at least passage 8. In some embodiments, the nMPCs are: (a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and (b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. Such nMPCs are non-naturally occurring. In some embodiments, the nMPCs are allogeneic.

[0055] In some embodiments, the nMPCs are negative for: (i) a mutation in, or modified expression of, a SOD1 gene or RNA; (ii) a mutation in, or modified expression of, chromosome 9, open reading frame 72, optionally wherein the chromosome 9, open reading frame 72 comprises a C9ORF72 gene; (iii) a fused Fus gene mutation; (iv) a mutation in, or modified expression of, a TDP-43 gene or RNA; (v) a mutation in, or modified expression of, a STMN2 gene or RNA; (vi) a mutation in, or modified expression of, a UNC13A gene or RNA; (vii) a mutation in, or modified expression of, a TARDBP gene or RNA; (viii) a mutation in, or modified expression of, hnRNPAl gene or RNA; (ix) a mutation in, or modified expression of, a hnRNPA2B 1 gene or RNA; (x) a mutation in, or modified expression of, a MATR3 gene or RNA; (xi) a mutation in, or modified expression of, an ANG gene or RNA; (xii) a mutation in, or modified expression of, a TUBA4A gene or RNA; (xiii) a mutation in, or modifiedWSGR Ref. No. : 64320-709.601 expression of, anANXAll gene or RNA; (xiv) a mutation in, or modified expression of, aPRPH gene or RNA; (xv) a mutation in, or modified expression of, a DCTN1 gene or RNA; (xvi) a mutation in, or modified expression of, aPFNl gene or RNA; (xvii) a mutation in, or modified expression of, a KIF5a gene or RNA; (xviii) a mutation in, or modified expression of, a UBQLN2 gene or RNA; (xix) a mutation in, or modified expression of, aSQSTMl gene or RNA; (xx) a mutation in, or modified expression of, an OPEN gene or RNA; (xxi) a mutation in, or modified expression of, a VCP gene or RNA; (xxii) a mutation in, or modified expression of, a CHMP2B gene or RNA; (xxiii) a mutation in, or modified expression of, a VAPB / VAMP gene or RNA; or (xxiv) a mutation in, or modified expression of, a TBK1 gene or RNA; (xxv) a mutation in, or modified expression of, an EphA4 gene or RNA; (xxvi) a mutation in, or modified expression of, a NEKA gene or RNA; (xxvii) a mutation in, or modified expression of, an ATXN2 gene or RNA. In some embodiments, a mutation includes an addition, a deletion, or a replacement of one or more nucleotides. Alternatively, or in addition, a mutation includes nucleotide repeats.

[0056] nMPCs described herein are isolated from the right atrial appendage of neonatal (<30 days old) donors with normal myocardium undergoing congenital heart surgery. The therapeutic benefits of nMPCs described herein can, in some instances, be exerted through their secretomes (conditioned media) and / or exosomes which comprise, for example, independently secreted growth factors, cytokines, chemokines, and / or miRNA. In some embodiments, the nMPCs are obtained from cardiac tissue from a neonate of less than 1 month (30 days) of age. In some embodiments, the neonatal donor is less than about 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, or 30 days old.

[0057] Neonatal cardiac mesenchymal progenitor cells (nMPCs) may be isolated from neonatal cardiac tissue by disaggregating tissue from the tissue of the right atrial appendage which is to serve as the source of the neonatal cardiac mesenchymal progenitor cells that are non-naturally occurring and subsequently cultured in vitro. For example, the tissue or organ can be disaggregated mechanically and / or treated with digestive enzymes and / or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, DNase, pronase, and / or dispase, etc. Mechanical disruption can also be accomplished by a number of methods including the use of grinders, blenders, sieves, homogenizers, pressure cells, sonicators, etc. In some embodiments, nMPCs are cultured under hypoxic conditions. In some embodiments, nMPCs may be cultured according to the disclosures in Example 1. The nMPCs as described and claimed herein are not naturally occurring as demonstrated by differences in the cell phenotype and secretome profile following the culture conditions.

[0058] Once the tissue has been reduced to a suspension, the suspension can be fractionated into subpopulations from which neonatal cardiac mesenchymal progenitor cells can be obtained. This also may be accomplished using techniques for cell separation including, but not limited to, cloning andWSGR Ref. No. : 64320-709.601 selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, standard and zonal centrifugation, centrifugal elutriation (counter-streaming centrifugation), unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cell sorting (FACS).

[0059] In one embodiment, nMPCs can be substantially free of cells that have marker profiles other than those associated with nMPCs described herein. In certain embodiments, the nMPCs are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% free of such contaminating cell types. In certain embodiments, the nMPCs are at least 80%, free of such contaminating cell types. In certain embodiments, the nMPCs are at least 85%, free of such contaminating cell types. In certain embodiments, the nMPCs are at least 90%, free of such contaminating cell types. In certain embodiments, the nMPCs are at least 95%, free of such contaminating cell types. Alternatively, or in addition, the nMPCs can also be substantially free of soluble, naturally occurring molecules. As discussed more fully below, a substantially purified progenitor cell of the disclosure can be obtained, for example, by extraction (e.g., via density gradient centrifugation and / or flow cytometry) from a culture source. Purity can be measured by any appropriate method. nMPCs described herein can be about 95%-100% purified by, for example, flow cytometry (e.g, FACS analysis), as discussed herein. Such purified nMPCs may, in certain instances, lack any retroviral DNA or retroviral RNA. In certain instances, an exemplary method of obtaining the nMPCs comprises the steps of: (a) culturing nMPCs in progenitor cell culture medium (e.g, ISTEM® from Progenitor Cells, Inc.); (b) treating the cells with a cell detachment solution (e.g, ACCUTASE® from Gibco # Al 1105- 05) and (c) collecting cells from the enzyme-treated culture to obtain the isolated nMPCs prior to one or more additional culturing methods. In some embodiments, the isolated nMPCs are allogeneic to a subject to be treated by the methods described herein.

[0060] In one embodiment, neonatal cardiac mesenchymal progenitor cell (nMPC) formulations comprise neonatal cardiac mesenchymal progenitor cells. In another embodiment, neonatal mesenchymal progenitor cell (nMPC) formulations consist of neonatal cardiac mesenchymal progenitor cells. In some embodiments, neonatal cardiac mesenchymal progenitor cell (nMPC) formulations comprise neonatal mesenchymal progenitor cells and nMPC secretomes. In other embodiments, neonatal cardiac mesenchymal progenitor cell (nMPC) formulations comprise neonatal mesenchymal progenitor cells and nMPC exosomes. In some embodiments, neonatal cardiac mesenchymal progenitor cell (nMPC) formulations comprise neonatal mesenchymal progenitor cell secretomes. In some embodiments, a formulation comprises cell-free neonatal cardiac mesenchymal progenitor cell secretomes (conditioned media). In some embodiments, neonatal cardiac mesenchymal progenitor cell (nMPC) formulations consist of cell-free neonatal cardiac mesenchymal progenitor cell exosomes. In certain embodiments, nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof are allogeneic.WSGR Ref. No. : 64320-709.601

[0061] In one embodiment, nMPCs are genetically engineered. Genetically engineering may encompass the introduction of a nucleic acid molecule (e.g, a plasmid) which encodes one or more paracrine factors. The paracrine factors may be exogenous or endogenous. In other embodiments, genetic engineering may encompass the introduction of an exogenous construct (e.g, a plasmid or an mRNA) that expresses a secreted growth factor, a cytokine, a chemokine, and / or a microRNA. In the case of an endogenous nucleic acid sequence, the nucleic acid molecule may contain a promoter and / or enhancer which induces expression or overexpression of a protein. In one non-limiting example, nMPCs are genetically engineered to express or overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. In some embodiments, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1. In another non-limiting example, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5, (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4, and are genetically engineered to express or overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. Alternatively, or in addition, nMPCs can be further negative for protein expression of p!6INK4aup to at least passage 8. In one embodiment, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2, (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and p!6INK4a, and (c) genetically engineered to express or overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1. In one instance, the nMPCs are genetically engineered to express or to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1. In another embodiment, the disclosure provides isolated nMPCs that have been transfected with an exogenous or recombinant construct (e.g., aplasmid) or transcript (e.g, an mRNA). In another non-limiting example, nMPCs are transfected with one or more exogenous or recombinant construct(s) or transcript(s) that cause(s) the nMPCs to express or overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF 1. In yet another non-limiting example, nMPCs are transfected with one or more exogenous or recombinant const ruct(s) or transcript(s) that cause(s) the nMPCs to express or overexpress one or more markers (e.g, proteins) provided in Table 4. In some embodiments, a neonatal cardiac mesenchymal progenitor cell is genetically modified to improve the protein folding or stability of a paracrine factor (e.g, HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1). In some embodiments, the conservative amino acid changes are made to a paracrine factor in an nMPC. In an embodiment, the conservative amino acid change improves the stability of a paracrine factor. In some embodiments, the conservative amino acid changes result in an increase in the levels of a paracrine factor. A transgene may be introduced into a neonatal cardiac mesenchymal progenitor cell via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran- mediated transfection, lipof ection, electroporation, or microinjection. By way of example only, a transgene may be introduced into cells using an appropriate expression vector including, but not limited to, cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses, andWSGR Ref. No.: 64320-709.601 adeno-associated viruses). Transfection can be obtained by using methods including culturing the cells on a monolayer of virus-producing cells.

[0062] The disclosure also provides methods of culturing the neonatal cardiac mesenchymal progenitor cell under conditions to promote the expression of selected coding sequences. In some embodiments, the coding sequence(s) can comprise a growth factor, a cytokine, a chemokine, and / or a microRNA. In one non-limiting example, the coding sequence(s) can encode HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1. In some embodiments, the cytokine may comprise an antiinflammatory cytokine (e.g., IL-1, IL4, IL-10, IL-13, TGF- , etc.). Expression includes, for example, miRNA and / or protein. In one embodiment, the cells are passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times. In another embodiment, the cells are cultured for at least 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 or more days. In yet another embodiment, the cells are cultured in conditioned media comprising a complete medium, such as, for example, ISTEM® medium.

[0063] The disclosure also provides nMPCs obtained by the methods described herein. In another embodiment, the nMPCs are substantially free of any retroviral DNA or RNA (e.g, 80% or greater retroviral DNA-free or retroviral RNA-free).

[0064] In certain embodiments, nMPCs express peptides, proteins, miRNA, or a combination thereof, downregulate an inflammatory, and / or an oxidative stress pathway (ERK / MAPK signaling pathway), while promoting cellular growth, survival, and / or proliferation pathways. In some embodiments, nMPCs operate through a multimodal mechanism of action, enhancing pathways related to, for example, angiogenesis, immunomodulation, energy production, and cardiomyocyte proliferation while reducing oxidative stress and / or inflammation. In some embodiments, nMPCs can significantly reduce the expression of fibrosis markers assessed and with superior potency compared to the other cell types, such as, for example, adult MPCs (e.g, adult counterpart of nMPCs, derived from adult right atrial appendage), bone marrow- derived MSCs (BM-MSCs), or normal human dermal fibroblasts. In other embodiments, nMPCs can positively modulate the immune system by, for example, significantly increasing Treg cell populations. In some embodiments, nMPCs can downregulate cytokines. In some instances, nMPCs can downregulate inflammatory cytokines. In some embodiments, the downregulated cytokines comprise pro-inflammatory cytokines (e.g., Interleukin (IL)- 1 [3, IL-6, IL-8, IL-12, IL-17a, IL- 18, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-a, interferon gamma (IFN-y)). In one embodiment, the downregulated cytokines comprise TNF-a, IL-8, MCP-1, IL-17a, and IFN-y. In another embodiment, the downregulated cytokines comprise TNF-a, IL-8, and MCP-1. In yet another embodiment, the downregulated cytokines comprise IL-17a and IFN-y. In an embodiment, nMPCs have higher immunomodulatory potential when compared to other clinically relevant cell types such as, for example, Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs), Cardiosphere- Derived Cells (CDCs), or aMPCs, as assessed by co-culturing cluster of differentiation (CD)4+T cells, and determining the proportion of CD4+, CD25+, and FoxP3+regulatory T cells (Tregs) after about 5, 6, 7, 8, 9, or 10 days by flow cytometry.WSGR Ref. No. : 64320-709.601

[0065] In some embodiments, nMPCs can express a microRNA (or “miRNA”). MicroRNAs are from about 19 to about 25 nucleotides long noncoding RNAs that bind to the 3' UTR of nucleic acid molecules and downregulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. A microRNA sequence includes a “seed” region, e.g., a sequence in the region of positions 2- 8 of the mature microRNA, which sequence has Watson-Crick complementarity to the miRNA target sequence. nMPC Secretomes (Conditioned Media) and Exosomes

[0066] In one aspect, provided herein are nMPC secretomes. An nMPC secretome (conditioned medium) can comprise nano- vesicles, microvesicles, exosomes, macromolecules such as peptides or proteins (e.g, growth factors, cytokines, etc.) and nucleic acids (e.g, miRNA, RNA, DNA), or a combination thereof. A non-limiting example of an nMPC secretome disclosed herein can comprise a chemokine, an interleukin, a growth factor, or any combination thereof. An nMPC secretome disclosed herein can comprise micro-vesicles, exosomes, or a combination thereof. In some aspects, the producer cell is a neonatal cardiac mesenchymal progenitor cell (nMPC). In some embodiments, an nMPC secretome or nMPC exosome as described herein is allogeneic.

[0067] In another non-limiting example, an nMPC secretome further comprises nMPC exosomes. In certain embodiments, an nMPC secretome is isolated or purified and is not naturally occurring in a host organism or progenitor cell, from which the secretome may be derived. In some instances, the nMPC secretome is purified or extracted from a progenitor cell (e.g., an nMPC) culture or medium. In some embodiments, the nMPC secretome is non-naturally occurring or engineered. In some embodiments, an nMPC secretome can comprise a microRNA. In certain embodiments, nMPCs can be genetically modified to upregulate or downregulate a desired cellular or secretome protein or peptide. In some instances, the secretome may contain one or more proteins comprising a cytokine, a chemokine, a growth factor, a soluble molecule, or a combination thereof. In some instances, the one or more proteins can be separate from exosomes or microparticles. In some instances, the one or more proteins can be on the surface of exosomes or microparticles. In some instances, the one or more proteins can be encapsulated by exosomes or microparticles. In some instances, the exosomes have an average particle diameter of for example, from about 20 nm to about 250 nm in diameter or from about 50 nm to about 100 nm in diameter. In some cases, a pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0068] As used herein the term “exosome” refers to a small (from about 20 nm to about 300 nm in diameter, from about 50 nm to about 200 nm in diameter, or from about 50 nm to about 100 nm in diameter) nMPC vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome is a species of extracellular vesicle. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g, a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g, a nucleic acid, miRNA, RNA, or DNA), a sugar (e.g, a simple sugar, polysaccharide, or glycan) or other molecules. The exosome is an nMPC exosome, and isolatedWSGR Ref. No.: 64320-709.601 from the nMPCs based on its size, density, biochemical parameters, or a combination thereof. Exosomes generally contain a lipid bilayer with a center containing fluid, DNA, RNA, miRNAs, peptides, and / or proteins. The bilayer comprises lipids including, but not limited to, cholesterol, a diglyceride, a ceramide, a sphingolipid (e.g., sphingomyelin, ceramide, etc.), a phospholipid, a glycerophospholipid (such as, for example, phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and polyglycerophospholipids (e.g, bisphosphate)), gangliosides (GM), or a combination thereof. The nMPC exosomes can also comprise a transmembrane protein, tetraspanin, antigen presenting molecule, glycoprotein, adhesion molecule, heat shock protein (Hsp), cytoskeletal protein, ESCRT component, membrane transport protein, fusion protein, growth factor, cytokine, lipid, fas ligand (FasL), mRNA, miRNA, non-coding RNA, DNA, TNF receptor, or transferrin receptor (TfR), or a combination thereof.

[0069] As used herein, the term “extracellular vesicle” or “EV” refers to a cell -derived vesicle comprising a membrane that encloses an internal space. Extracellular vesicles comprise all membranebound vesicles that have a smaller diameter than the cell from which they are derived. Generally, extracellular vesicles can range in diameter from about 20 nm to about 1000 nm and can comprise various macromolecular payload either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. In certain embodiments, extracellular vesicles range in diameter from about 50 nm to about 200 nm and can comprise various macromolecular payloads either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. Said payload can comprise nucleic acids (e.g., DNA, RNA, miRNA, etc.), peptides, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. RNA can comprise, for example, miRNAs, messenger mRNA (mRNA), circular RNA (circRNA), IncRNA (long non-coding RNA), etc. By way of example and without limitation, extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g, by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g, by direct plasma membrane budding or fusion of the late endosome with the plasma membrane).Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and / or cultured cells. In some embodiments, an nMPC exosome, as described herein, is allogeneic.

[0070] In certain embodiments, an nMPC secretome comprises peptides, proteins, miRNA, or a combination thereof, responsible for downregulating a fibrotic, inflammatory, and / or oxidative stress pathway (ERK / MAPK signaling pathway), while promoting cellular growth, survival, and / or proliferation pathways. In some embodiments, an nMPC secretome operates through a multimodal mechanism of action, enhancing pathways related to, for example, angiogenesis, immunomodulation, energy production, and cardiomyocyte proliferation while reducing oxidative stress, inflammation, and / or fibrosis. In some embodiments, an nMPC secretome can significantly reduce the expression of fibrosis markers assessed and with superior potency compared to the other cell types, such as, for example, adult MSCs (e.g., adult counterpart of nMPCs, derived from an adult right atrial appendage), bone marrow- derived MSCs (BM-MSCs), or normal human dermal fibroblasts. In other embodiments, an nMPCWSGR Ref. No. : 64320-709.601 secretome can positively modulate the immune system by, for example, significantly increasing Treg cell population. In some embodiments, an nMPC secretome downregulates cytokines. In some embodiments, the downregulated cytokines comprise pro-inflammatory cytokines (e.g, Interleukin (IL)- ip, IL-6, IL-8, IL-12, IL-17a, IL-18, MCP-1, tumor necrosis factor (TNF)-a, interferon gamma (IFN-y)). In one embodiment, the downregulated cytokines comprise TNF-a, IL-8, MCP-1, IL-17a, and IFN-y. In another embodiment, the downregulated cytokines comprise TNF-a, IL-8, and MCP-1. In yet another embodiment, the downregulated cytokines comprise IL- 17a and IFN-y. In an embodiment, an nMPC secretome has higher immunomodulatory potential when compared to other clinically relevant cell types such as, for example, Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs), Cardiosphere- Derived Cells (CDCs), or aMPCs, as assessed by co-culturing cluster of differentiation (CD)4+T cells, and determining the proportion of CD4+, CD25+, and FoxP3+regulatory T cells (Tregs) after about 5, 6, 7, 8, 9, or 10 days by flow cytometry.

[0071] In one embodiment, the nMPC secretome or the nMPC exosome is isolated. In certain embodiments, the nMPC secretomes and / or nMPC exosomes are at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% free of cells. In one embodiment, the nMPC secretomes and / or nMPC exosomes are at least about 90% free of cells. In another embodiment, the nMPC secretomes and / or nMPC exosomes are at least about 95% free of cells. In one embodiment, the nMPC secretomes and / or nMPC exosomes are at least about 99% free of cells. In yet another embodiment, the nMPC secretomes and / or nMPC exosomes are at least about 99.5% free of cells.

[0072] Substantially purified nMPCs, nMPC secretome, and / or nMPC exosomes described herein can be obtained, for example, by extraction (e.g, via density gradient centrifugation and / or flow cytometry) and filtration (e.g. , size exclusion chromatography) from a culture source. Purity can be measured by any appropriate method.

[0073] A neonatal cardiac mesenchymal progenitor cell (nMPC) secretome and / or exosome of the disclosure can be about 99%-100% purified by, for example, size exclusion chromatography, as discussed herein. Such purified nMPC secretomes and / or nMPC exosomes may also lack any retroviral DNA or retroviral RNA. In certain instances, the method of obtaining the nMPC secretomes and / or exosomes comprise the steps of: (a) culturing nMPCs in progenitor cell culture medium (e.g, ISTEM® from Progenitor Cells, Inc., Rooster Basal™-MSC-CC, Rooster Booster™-MSC-CC, etc.); (b) treating the cells with a cell detachment solution (e.g., ACCUTASE® from Gibco # Al 1105-05, TrypLE™ Express Enzymes), (c) centrifuging the cultures by standard or ultracentrifugation, (d) collecting the supernatant, and optionally (e) using size exclusion chromatography. In some embodiments, an isolated or purified secretome and / or exosome as described herein is allogeneic. In some aspects, disclosed herein is a composition that comprises a secretome, e.g, a protein, an exosome, or a microvesicle secreted from a cell or cells (e.g, neonatal cardiac mesenchymal progenitor cells).

[0074] In some instances, the secretome is isolated or purified and is not naturally occurring in a host organism or progenitor cell, from which the secretome may be derived. In some instances, the secretome is purified or extracted from a progenitor cell (e.g., an nMPC) culture or medium. In some instances, theWSGR Ref. No. : 64320-709.601 secretome may contain one or more proteins comprising a cytokine, a chemokine, a growth factor, a soluble molecule, or any combination thereof. In some instances, the one or more proteins can be separate from exosomes or microparticles. In some instances, the one or more proteins can be on the surface of exosomes or microparticles. In some instances, the one or more proteins can be encapsulated by exosomes or microparticles. In some instances, the exosomes have an average particle diameter of for example, from about 20 nm to about 250 nm in diameter or from about 50 nm to about 100 nm in diameter. In some cases, a pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0075] In some embodiments, an nMPC secretome and / or nMPC exosomes are (a) positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, HSF1, and / or Akt, and (b) positive for miRNA expression of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, and / or miR-374b-5p. In one embodiment, the nMPC secretome and / or nMPC exosomes are (a) positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, HSF1, or Akt, and (b) positive for miRNA expression of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, or miR-374b-5p. In another embodiment, the nMPC secretome and / or nMPC exosomes are (a) positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, HSF1, and Akt, and (b) positive for miRNA expression of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, and miR-374b-5p. In certain embodiments, the nMPC secretome is positive for miRNA expression of one, two, three, four, five, or six of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, or miR- 374b-5p.

[0076] In some embodiments, the nMPC exosomes are positive for miRNA expression of one, two, three, four, five, or six of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, and miR-374b- 5p. In some embodiments, the nMPC exosomes are positive for protein expression of CD81 and / or CD63. In some embodiments, the nMPC exosomes are positive for protein expression of CD81 and CD63.

[0077] In some embodiments, an nMPC is genetically modified to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1 such that the level of each overexpressed factor is greater in the isolated nMPC secretome and / or nMPC exosomes as compared to the level of each factor in a secretome or exosomes isolated from nMPCs that have not been genetically modified. In some embodiments, an nMPC is genetically modified to overexpress miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, and / or miR-374b-5p such that the level of each overexpressed miRNA is greater in the isolated nMPC secretome and / or nMPC exosomes as compared to the level of each miRNA in a secretome isolated from nMPCs that have not been genetically modified.

[0078] In other embodiments, the nMPC secretome and / or nMPC exosomes have a marker (e.g., protein) expression profile as provided in Table 4. In some embodiments, an nMPC secretome and / or nMPC exosomes are negative for protein expression of IGF 1. In other embodiments, nMPCs are genetically engineered to overexpress one or more paracrine factors. In other embodiments, nMPCs are genetically engineered to overexpress one or more markers (e.g, proteins) provided in Table 4. In certainWSGR Ref. No. : 64320-709.601 embodiments, nMPCs can be genetically modified to upregulate or downregulate a desired cellular or exosomal protein or peptide that can be detected in the secretome by conventional means. In some embodiments, the nMPC secretome and / or nMPC exosomes are produced by culturing nMPCs cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days and collecting the supernatant of the cultured cells. In some embodiments, the nMPC secretome and / or nMPC exosomes are concentrated.

[0079] The nMPC exosomes can be characterized by their particle size. In some embodiments, an exosome described herein can comprise a particle in a range of from about 50 nm to about 100 nm, from about 50 nm to about 190 nm, from about 50 nm to about 180 nm, from about 50 nm to about 170 nm, from about 50 nm to about 160 nm, from about 50 nm to about 150 nm, from about 50 nm to about 140 nm, from about 50 nm to about 130 nm, from about 50 nm to about 120 nm, from about 50 nm to about 110 nm, from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, or from about 50 nm to about 60 nm in diameter. In an embodiment, an exosome described herein can comprise a particle from about 50 nm to about 90 nm in diameter. In an embodiment, an exosome described herein can comprise a particle from about 50 nm to about 100 nm in diameter. In an embodiment, an exosome described herein can comprise a particle from about 50 nmto about 110 nm in diameter. The nMPC exosomes described herein can comprise particles in a range of from about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 900 nm, about 700 nm to about 800 nm, about 300 nm to about 600 nm, about 300 nm to about 500 nm, 300 nm to about 400 nm, about 200 nm to about 500 nm, 200 nm to about 400 nm, about 100 nm to about 500 nm, about 100 nm to about 300 nm, 100 nm to about 200 nm, 100 nm to about 150 nm, about 50 nm to about 200 nm, about 50 nmto about 150 nm, about 50 nm to about 110 nm, about 50 nm to about 100 nm, about 75 nm to about 250 nm, about 75 nm to about 200 nm, about 75 nm to about 150 nm, about 75 nm to about 100 nm, about 90 nm to about 150 nm, about 90 nm to about 125 nm, about 90 nm to about 110 nm, or about 90 nm to about 100 nm in diameter. In one embodiment, the nMPC exosomes comprise particles in a range of from about 90 nm to about 110 nm in diameter. In another embodiment, the nMPC exosomes comprise particles in a range of from about 100 nm to about 120 nm in diameter. In some embodiments, the nMPC exosomes comprise particles about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm in diameter. In some embodiments, the nMPC exosomes comprise particles about 90 nm in diameter. In certain embodiments, the nMPC exosomes comprise particles about 100 nm in diameter. In other embodiments, the nMPC exosomes comprise particles about 110 nm in diameter. In yet other embodiments, the nMPC exosomes have a density of about 1.13 g / ml to about 1.19 g / ml.

[0080] In certain embodiments, an nMPC secretome comprises peptides, proteins, miRNA, or a combination thereof, responsible for downregulating a fibrotic, inflammatory, and / or oxidative stress pathway (ERK / MAPK signaling pathway), while promoting cellular growth, survival, and / orWSGR Ref. No. : 64320-709.601 proliferation pathways. In some embodiments, an nMPC secretome operates through a multimodal mechanism of action, enhancing pathways related to, for example, angiogenesis, immunomodulation, energy production, and cardiomyocyte proliferation while reducing oxidative stress, inflammation, and / or fibrosis. In some embodiments, an nMPC secretome can significantly reduce the expression of fibrosis markers assessed and with superior potency compared to the other cell types, such as, for example, adult MPCs (e.g., adult counterpart of nMPCs, derived from an adult right atrial appendage), bone marrow- derived MSCs (BM-MSCs), or normal human dermal fibroblasts. In other embodiments, an nMPC secretome can positively modulate the immune system by, for example, significantly increasing Treg cell populations. In some embodiments, an nMPC secretome downregulates cytokines. In some embodiments, the downregulated cytokines comprise pro-inflammatory cytokines (e.g., Interleukin (IL)- ip, IL-6, IL-8, IL-12, IL-17a, IL-18, MCP-1, tumor necrosis factor (TNF)-a, interferon gamma (IFN-y)). In one embodiment, the downregulated cytokines comprise TNF-a, IL-8, MCP-1, IL-17a, and IFN-y. In another embodiment, the downregulated cytokines comprise TNF-a, IL-8, and MCP-1. In yet another embodiment, the downregulated cytokines comprise IL- 17a and IFN-y. In an embodiment, an nMPC secretome has higher immunomodulatory potential when compared to other clinically relevant cell types such as, for example, BM-MSCs, CDCs, or aMPCs, as assessed by co-culturing cluster of differentiation (CD)4+T cells, and determining the proportion of CD4+, CD25+, and FoxP3+regulatory T cells (Tregs) after about 5, 6, 7, 8, 9, or 10 days by flow cytometry.

[0081] In some embodiments, the nMPC secretome is non-naturally occurring or engineered. In some embodiments, an nMPC secretome can comprise a microRNA. In certain embodiments, nMPCs can be genetically modified to upregulate or downregulate a desired cellular or secretome protein or peptide. In some embodiments an nMPC secretome is positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and / or Akt. In some embodiments, the nMPC secretome comprises HSF1 at levels higher than HSF1 in the secretome of aMPCs. In some embodiments an nMPC is genetically modified to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1 such that the level of each overexpressed factor is greater in the isolated nMPC secretome as compared to the level of each factor in a secretome isolated from nMPCs that have not been genetically modified. In one instance, the secretome is positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and Akt. In other embodiments, the nMPC secretome has a marker (e.g, protein) expression profile as provided in Table 4. In some embodiments an nMPC secretome is positive for protein expression of IGF 1. In other embodiments, nMPCs are genetically engineered to overexpress one or more paracrine factors. In other embodiments, nMPCs are genetically engineered to overexpress one or more markers (e.g., proteins) provided in Table 4. In certain embodiments, nMPCs can be genetically modified to upregulate or downregulate a desired cellular or exosomal protein or peptide that can be detected in the secretome by conventional means. In some embodiments, the secretome is produced by culturing nMPCs cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days and collecting the supernatant of the cultured cells. In some embodiments, the nMPC secretome is concentrated.WSGR Ref. No. : 64320-709.601Compositions and Formulations

[0082] The terms “pharmaceutical composition” and “pharmaceutical formulation” (or “formulation”) are used interchangeably and denote a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients to be administered to a subject, e.g, a human in need thereof. A pharmaceutical composition used in the therapeutic methods of the invention is formulated to be compatible with its intended route of administration. In certain embodiments, the active pharmaceutical ingredient comprises, for example, neonatal cardiac mesenchymal progenitor cells, nMPC secretomes (nMPC conditioned media), nMPC exosomes, nMPC total secretome, or any combination thereof. In some embodiments, the pharmaceutical formulation comprises nMPCs only in a pharmaceutically acceptable carrier (e.g, phosphate buffered saline). In some embodiments, the pharmaceutical formulation comprises nMPCs and nMPC secretome (e.g., conditioned media) in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises nMPC secretome (e.g, conditioned media) in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises nMPC exosomes only in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises nMPCs, nMPC secretomes, nMPC exosomes, peptides or proteins (e.g., cytokines, growth factors, etc. ), nucleic acids (e.g, miRNA, mRNA, DNA), or any combination thereof. In one embodiment, the pharmaceutical formulation comprises nMPCs, nMPC secretomes (comprising, for example, peptides and / or proteins (e.g, growth factors, cytokines), and nucleic acids (e.g, miRNA, mRNA, DNA)), nMPC exosomes, nMPC total secretome, or a combination thereof in a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical formulation disclosed herein is administered in combination with a drug or therapeutic agent. In certain aspects, combination therapy is contemplated wherein the nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof are administered with a drug or a therapeutic agent. For example, in some embodiments, the drug or therapeutic agent comprises Riluzole, Edaravone, Tofersen, Nuedexta, sodium phenylbutyrate- taurursodiol (PB-TUDCA), or a combination thereof.

[0083] In one embodiment, the pharmaceutical composition is serum-free, protein-free, sterile, free of toxins, GMP certified, free of endotoxins, or free of pathogens.

[0084] In some embodiments, an IV bag comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 doses of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof. In certain embodiments, an IV bag comprises a single dose of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof. In one instance, an IV bag comprises 4 doses of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof. In another instance, an IV bag comprises 7 doses of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof. In some embodiments, a dose of nMPCs is administered at a range of from about 200,000 cells / 100 pl to about IxlO6cells Z100 pl.WSGR Ref. No. : 64320-709.601

[0085] In some embodiments, an IV bag comprises a total of from about 50 million to about 250 million cells. In some embodiments, an IV bag comprises a total of about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, or about 250 million cells. In some embodiments, an IV bag comprises a total of about 50 million cells. In some embodiments, an IV bag comprises a total of about 100 million cells. In some embodiments, an IV bag comprises a total of about 150 million cells. In some embodiments, an IV bag comprises a total of about 200 million cells. In some embodiments, an IV bag comprises a total of about 250 million cells. In some embodiments, a human subject is administered with one or more IV bags comprising nMPCs. In some embodiments, when more than one IV bag is administered, each bag is administered at about every 28 days.Protein Concentrations

[0086] The compositions described herein can comprise the nMPC secretome and / or nMPC exosomes at a final protein concentration. A final protein concentration can be adjusted to achieve therapeutic efficacy depending on measured symptoms of a subject by a clinician. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of from about 0. 1 mg / ml to about 50.0 mg / ml, or from about 0.5 mg / ml to about 20.0 mg / ml. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of (a) at least about 0.001 mg / ml, 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, about 2.0 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, about 11.0 mg / ml, about 12.0 mg / ml, about 13.0 mg / ml, about 14.0 mg / ml, about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 18.0 mg / ml, about 19.0 mg / ml, about 20.0 mg / ml, about 22.0 mg / ml, about 24.0 mg / ml, about 26.0 mg / ml, about 28.0 mg / ml, about 30.0 mg / ml, about 35.0 mg / ml, about 40.0 mg / ml, about 45.0 mg / ml, or about 50.0 mg / ml, or (b) at most about 250.0 mg / ml, about 225.0 mg / ml, about 200.0 mg / ml, about 175 mg / ml, about 150 mg / ml, about 125 mg / ml, about 100 mg / ml, about 75 mg / ml, about 50 mg / ml, about 45.0 mg / ml, about 40.0 mg / ml, about 35.0 mg / ml, about 30.0 mg / ml, about 25 mg / ml, about 20 mg / ml, about 15 mg / ml, about 10 mg / ml, about 5 mg / ml, about 1 mg / ml, about 0.5 mg / ml, about 0.1 mg / ml, about 0.01 mg / ml, or about 0.001 mg / ml. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 0.001 mg / ml ±0.001, ±0.002, ±0.003, ±0.004, or ±0.005. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 0.01 mg / ml ±0.01, ±0.02, ±0.03, ±0.04, or ±0.05. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 1.0 mg / ml ±0.1, ±0.2, ±0.3, ±0.4, or ±0.5. In other embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 4.0 mg / ml ±0.5, ±1.0, ±1.5, ±2.0, ±2.5, or ±3.0. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 8.0 mg / ml ±1.0, ±2.0, ±3.0, ±4.0, or ±5.0. In certain embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 16.0 mg / ml ±2.0, ±3.0, ±4.0, ±5.0, or ±6.0. In some instances, the nMPC secretome and / or nMPC exosomes are administered at aWSGR Ref. No. : 64320-709.601 concentration of at least about 10 pg / ml to at least about 1000 pg / ml. Alternatively, or in addition, in certain embodiments, the nMPC secretome and / or nMPC exosomes are positive for protein expression of one or more of the proteins in Table 4.

[0087] The separate components of the compositions or formulations disclosed herein may be preblended or each component may be added separately to the same environment according to a predetermined dosage for the purpose of achieving the desired concentration level of the treatment components and so long as the components eventually come into intimate admixture with each other. Further, the compositions or formulations disclosed herein may be administered or delivered on a continuous or intermittent basis. In some embodiments, nMPC secretomes and / or nMPC exosomes may be separately produced, isolated, and concentrated, followed by the blending or titration of a specific number or concentration of nMPCs into a volume of the composition or formulation comprising nMPC secretomes and / or nMPC exosomes.PH

[0088] The maintenance of a biologically suitable pH is also contemplated for the compositions described herein. In certain embodiments, the composition comprises a pH of from about 5.2 to about 7.7, from about 5.5 to about 7.5, from about 7.1 to about 7.5, from about 7.2 to about 7.4, from about 6.0 to about 8.0, from about 7.0 to about 9.0, or from about 5.0 to about 9.0. In one embodiment, the composition comprises a pH of about 6.0. In one embodiment, the composition comprises a pH of about 6.2. In one embodiment, the composition comprises a pH of about 6.5. In one embodiment, the composition comprises a pH of about 7.0. In another embodiment, the composition comprises a pH of about 7.2. In one embodiment, the composition comprises a pH of 7.4. In some embodiments, the composition comprises a pH of about 7.5. In certain other embodiments, the composition comprises a pH of about 7.7. In one embodiment, the composition comprises a pH of about 8.5. In another embodiment, the composition comprises a pH of about 8.9. In some embodiments, the pH of a composition upon administration is the pH at a normal body temperature range from about 97 °F to about 99 °F (about 36. 1 °C to about 37.2 °C). In certain other embodiments, the pH of a composition prior to administration and / or in storage comprises a pH of from about 5.0 to about 8.0, from about 6.0 to about 8.0, from about 7.0 to about 9.0, or from about 5.0 to about 9.0. In some embodiments, the pH of a composition described herein is the pH as measured at about 4 °C, 10 °C, 25 °C, or about 37 °C. In one embodiment, the pH of a composition is between about 6.8 and about 7.9 at about 25 °C. In one embodiment, the pH of a composition is about 7.4 at about 25 °C. In another embodiment, the pH of a composition is between about 6.8 and about 7.9 at about 37 °C.Lyophilization

[0089] Lyophilization, also known as freeze-drying, is a preservation technique that removes water from a material, typically by freezing and then sublimating the ice into vapor under a vacuum. This process can help stabilizing sensitive materials like pharmaceuticals, biological samples, to extend their shelf life and improve ease of transportation and storage. Lyophilization of the compositions described herein is also contemplated, and may be useful for environments or situations in which a patient may have limitedWSGR Ref. No. : 64320-709.601 access to modem medical facilities. In some embodiments, the composition is lyophilized. In certain embodiments, the lyophilized composition is reconstituted in a diluent. In certain embodiments, the diluent comprises a pharmaceutically acceptable diluent. In other embodiments, the lyophilized composition is incubated for an amount of time and / or at a suitable temperature comprising, for example, a temperature of about 4 °C, 10 °C, 25 °C, or about 37 °C.Pharmaceutically Acceptable Excipients

[0090] In any such compositions, a pharmaceutically acceptable carrier may comprise, for example, buffers, surfactants, stabilizers, preservatives, suspending agents, thickening agents, viscosity regulators, dispersion media, coatings, antibacterial agents, antifungal agents, cryoprotectants isotonic and absorption delaying agents, and osmoregulators. In some instances, a pharmaceutically acceptable carrier may comprise physiologically relevant phosphate buffered saline (e.g, 0.9% sterile PBS at a pH of about 7.4).Considerations for Intravenous Administration

[0091] Formulations for intravenous injection are also contemplated. Compositions can comprise the nMPCs, and in such cases, dosage units may comprise the number of cells to be administered in each dose. In some embodiments, a dose of the nMPCs comprises from about 15 million to about 250 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 35 million to about 65 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 45 million to about 55 million nMPCs. In some embodiments, a dose of the nMPCs comprises about 50 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 85 million to about 115 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 95 million to about 105 million nMPCs. In some embodiments, a dose of the nMPCs comprises about 100 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 135 million to about 165 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 145 million to about 155 million nMPCs. In some embodiments, a dose of the nMPCs comprises about 150 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 185 million to about 215 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 195 million to about 205 million nMPCs. In some embodiments, a dose of the nMPCs comprises about 200 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 235 million to about 265 million nMPCs. In some embodiments, a dose of the nMPCs comprises from about 245 million to about 255 million nMPCs. In some embodiments, a dose of the nMPCs comprises about 250 million nMPCs. In some embodiments, the subject is administered an intravenous (IV) bag comprising a total of from about 50 million to about 250 million nMPCs.

[0092] Compositions for intravenous administration can also comprise nMPC secretomes and / or nMPC exosomes. In such cases, a dosage unit may comprise the final protein concentration in a dose, as calculated from the total weight of protein in the nMPC secretomes and / or nMPC exosomes per volume (for example, in milliliters) administered to the subject. A final protein concentration can be adjusted to achieve therapeutic efficacy depending on measured symptoms of a subject by a clinician. In someWSGR Ref. No. : 64320-709.601 embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of from about 0. 1 mg / ml to about 50.0 mg / ml, or from about 0.5 mg / ml to about 20.0 mg / ml. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of (a) at least about 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, about 2.0 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, about 11.0 mg / ml, about 12.0 mg / ml, about 13.0 mg / ml, about 14.0 mg / ml, about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 18.0 mg / ml, about 19.0 mg / ml, about 20.0 mg / ml, about 22.0 mg / ml, about 24.0 mg / ml, about 26.0 mg / ml, about 28.0 mg / ml, about 30.0 mg / ml, about 35.0 mg / ml, about 40.0 mg / ml, about 45.0 mg / ml, or about 50.0 mg / ml, or (b) at most about 250.0 mg / ml, about 225.0 mg / ml, about 200.0 mg / ml, about 175 mg / ml, about 150 mg / ml, about 125 mg / ml, about 100 mg / ml, about 75 mg / ml, about 50 mg / ml, about 45.0 mg / ml, about 40.0 mg / ml, about 35.0 mg / ml, about 30.0 mg / ml, about 25 mg / ml, about 20 mg / ml, about 15 mg / ml, about 10 mg / ml, about 5 mg / ml, about 1 mg / ml, about 0.5 mg / ml, or about 0.1 mg / ml. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 1.0 mg / ml ±0.1, ±0.2, ±0.3, ±0.4, or ±0.5. In other embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 4.0 mg / ml ±0.5, ±1.0, ±1.5, ±2.0, ±2.5, or ±3.0. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 8.0 mg / ml ±1.0, ±2.0, ±3.0, ±4.0, or ±5.0. In certain embodiments, the nMPC secretome and / or nMPC exosomes comprise a final protein concentration of at least about 16.0 mg / ml ±2.0, ±3.0, ±4.0, ±5.0, or ±6.0. In some embodiments, a dosage may be calculated by the total protein concentration of the composition to be administered per kilogram of body weight of the subject. In some embodiments, the subject is administered the nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof at a dosage of at least about 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 22.5 mg / kg, 25 mg / kg, 27.5 mg / kg, 30 mg / kg, 32.5 mg / kg, 35 mg / kg, 37.5 mg / kg, 40 mg / kg, 42.5 mg / kg, 45 mg / kg, 47.5 mg / kg, or 50 mg / kg, where mg / kg is the nMPC secretome or nMPC exosome protein weight per kilogram of subject body weight.

[0093] In some embodiments, provided herein are pharmaceutical compositions and methods of producing the pharmaceutical formulations. In any of such embodiments, nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof, may be introduced into a volume appropriate for intravenous infusion; for example, in an amount of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ml, etc. In the compositions disclosed herein, each dose comprising nMPCs, an nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof, comprises a volume of about a liter or less, about 500 ml or less, about 250 ml or less, or about 100 ml or less. In some embodiments, compositions or formulations comprising nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof, the compositions or formulations comprise a volume of a liter or less, about 500 ml or less, about 250 ml or less, or aboutWSGR Ref. No. : 64320-709.601100 ml or less for each dose. In certain embodiments, a composition or formulation as disclosed herein is manufactured in accordance with Good Manufacturing Practices (GMP), a set of quality assurance standards for the production of medicinal products.Storage Conditions

[0094] In any of such methods, the nMPCs are stable in storage at a temperature for a period of time. In some embodiments, the temperature is about 4 °C, 1 °C, 0 °C, -10 °C, -20 °C, or -80 °C. In some embodiments, the period of time is at least about 1, 2, 3, 4, 5, 6, or 7 days; at least about 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; or at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months. In some embodiments, the nMPCs are stored in cell cryopreservation media- CS10 (CRYOSTOR®; Sigma- Aldrich, Israel). In some embodiments, the nMPCs are thawed in a 37 °C water bath until a single ice crystal remained prior to administration to the subject. In some embodiment^ the nMPCs are further washed with a volume of Complete Medium (CM). In some embodiments, the nMPCs are washed twice with a volume of Complete Medium (CM). In some embodiments, the volume is at least about 10 ml. In some embodiments, the cryopreservation medium comprises a permeating cryoprotective agent. In some embodiments, the permeating cryoprotective agent comprises a US Pharmacopeia (USP)-grade permeating cryoprotective agent. In some embodiments, the cry opreservation medium comprises at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the permeating cryoprotective agent. In some embodiments, the cry opreservation medium comprises about 10% of the permeating cryoprotective agent. In some embodiments, the cry opreservation medium comprises at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of a Dimethyl Sulfoxide (DMSO). In some instances, the cry opreservation medium comprises about 10% of a Dimethyl Sulfoxide (DMSO).Protein Expression Detection

[0095] Assessment of the presence or levels of protein expression of the one or more factors (e.g, c-kit (CD117), HSF1, Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4, etc.) can be performed by, for example, detection of a signal (e.g, signal produced by a fluorescent protein) using FACS, or selection by another marker such as an antibiotic resistance marker. In some cases, the signal used to assess presence or levels of protein expression was produced by a fluorescently labeled antibody (e.g, an antibody labeled with FITC, phycoerythrin (PE), Peridinin-Chlorophyll-protein (PerCP), or Allophycocyanin (APC)) that could bind to a factor. In some cases, the presence or levels of protein expression of the one or more factors was measured by antibody staining, fluorescence microscopy, flow cytometry as discussed above, protein labeling, an enzyme-linked immunosorbent assay (ELISA), mass spectroscopy, western blotting, or Bradford protein assays. Such assays can be used to evaluate the level of expression of a factor: for example, higher protein expression levels may be correlated with stronger signals (e.g, fluorescent signals), and lower expression levels may be correlated with weaker signals. The cells may be separatedWSGR Ref. No. : 64320-709.601 based on the level of staining for a particular antigen or lack thereof. Expression levels are compared to a positive control and / or a negative control.RNA Sequencing

[0096] High-throughput RNA sequencing (RNA-Seq) technology, enabled by the developments in nextgeneration sequencing, represents a powerful tool in analyzing gene expression profiles, detecting transcript variants, and understanding the function of the non-coding regulatory RNAs. An RNA-Seq library can be generated from ligating sequencing adapters to double-stranded DNA. A first RNA-Seq library method of generating a comprises ligating different adapters to the 3' and 5' ends of the RNA molecules (see, e.g, Ion Total RNA-Seq Kit v2 from Life Technologies). A second method comprises incorporating dUTP in addition to dNTPs in the second strand DNA synthesis. Following adapter ligation, the second strand DNA can be specifically digested by an Uracil-N-glycosylase (UNG) enzyme so that only the library strand containing the first strand cDNA is sequenced and information on the direction of the transcripts can therefore be obtained (see, M. Sultan et al. , Biochemical and Biophysical Research Communications, 422 (2012) 643-646). A third method uses the techniques described in, for example, U. S. Pat. No. 11248262B2 in which a method of RNA sequencing comprises: (i) providing RNA; (ii) generating (a) single-stranded first DNA strand(s) (cDNA), which is / are complementary to the RNA, by subjecting the RNA to reverse transcription by using a reverse transcriptase, a first set of oligonucleotide primers, and the RNA of step (i), and (iii) generating a second DNA strand by using a DNA polymerase, a second set of oligonucleotide primers, and the single-stranded cDNA of (ii), wherein a) the first set of oligonucleotide primers comprises a covalently coupled moiety at its / their 5' terminal nucleotide, which blocks ligation at the 5' terminus of the generated first DNA strand; or b) the second set of oligonucleotide primers comprises a covalently coupled moiety at its / their 5' terminal nucleotide, which blocks ligation at the 5' terminus of the generated second DNA strand. In certain instances, the method further comprises the subsequent steps of: (iv) optionally end-repairing the double-stranded DNA strands using a polynucleotide kinase and an enzyme with polymerase and exonuclease activities to obtain end- repaired DNA strands; (v) optionally adding a terminal adenine to the 3' termini of the DNA strands using a deoxynucleotidyl transferase enzyme; and (vi) ligation of adapters, which optionally comprise terminal thymines, to the DNA strands, which optionally comprise 3' terminal adenines. The methods may further comprise sequence analysis of the generated DNA.

[0097] In some embodiments, said method comprises: (i) providing RNA; (ii) generating (a) singlestranded first DNA strand(s) (cDNA), which is / are complementary to the RNA, by subjecting the RNA to reverse transcription by using a reverse transcriptase, a first set of oligonucleotide primers, and the RNA of step (i); (iii) generating a second DNA strand by using a DNA polymerase, a second set of oligonucleotide primers, and the single-stranded cDNA of (ii); (iv) ligating adapters to the doublestranded DNA; of step (iii) and (v) sequencing the generated DNA, wherein a) the first set of oligonucleotide primers comprises a covalently coupled moiety at its / their 5' terminal nucleotide, which blocks ligation at the 5' terminus of the generated first DNA strand; or b) the second set of oligonucleotide primers comprises a covalently coupled moiety at its / their 5' terminal nucleotide, whichWSGR Ref. No. : 64320-709.601 blocks ligation at the 5' terminus of the generated second DNA strand. By generating the second DNA strand, a double-stranded DNA is generated.

[0098] In some embodiments of the above-mentioned method, prior to step (iv), the method comprises the step of: (iii)(a) end-repairing the double-stranded DNA strands using a polynucleotide kinase and an enzyme with polymerase and exonuclease activities to obtain end-repaired DNA strands.

[0099] In some embodiments, step (iii)(a) is followed by step (iii)(b) comprising adding a terminal adenine to the 3' termini of the DNA strands by using a deoxynucleotidyl transferase enzyme, wherein the adapters comprise 3' terminal thymines, which in step (iv) ligate to the DNA strands comprising 3' terminal adenines. In some embodiments, the oligonucleotide primers, which are covalently coupled to a blocking moiety and / or unmodified oligonucleotide primers, are random oligonucleotide primers.

[0100] In some embodiments, said methods comprise the initial step of extracting and optionally enriching the RNA of interest. In some embodiments, the extracted RNA is fragmented to an average size of from about 19 to about 510 bp. In some embodiments of the above methods, the molecules may be attached to a solid support for paired-end sequencing.Methods of Treatment

[0101] Neonatal cardiac mesenchymal progenitor cells, nMPC secretomes, and nMPC exomes described herein can be used to reduce the likelihood or severity of, inhibit, or treat one or more symptoms of ALS.

[0102] Subjects to be treated by the methods described herein are diagnosed as having ALS. Diagnosis can be measured through a comprehensive evaluation by a neurologist, including, for example, a thorough physical examination, medical history review, and various diagnostic tests (e.g., electromyography (EMG), nerve conduction studies, blood and urine tests, an MRI scan, etc.).

[0103] Subjects to be treated can be from about 4 to about 18 years of age, from about 19 to about 25 years of age, or 26 years of age or older. In one embodiment, the subject to be treated is about 4 years of age. In another embodiment, the subject to be treated is about 5 years of age. In another embodiment, the subject to be treated is about 6 years of age. In another embodiment, the subject to be treated is about 7 years of age. In another embodiment, the subject to be treated is about 8 years of age. In another embodiment, the subject to be treated is about 9 years of age. In another embodiment, the subject to be treated is about 10 years of age. In another embodiment, the subject to be treated is about 11 years of age. In another embodiment, the subject to be treated is about 12 years of age. In another embodiment, the subject to be treated is about 13 years of age. In another embodiment, the subject to be treated is about 14 years of age. In another embodiment, the subject to be treated is about 15 years of age. In another embodiment, the subject to be treated is about 16 years of age. In another embodiment, the subject to be treated is about 17 years of age. In another embodiment, the subj ect to be treated is about 18 years of age. In another embodiment, the subject to be treated is about 19 years of age. In another embodiment, the subject to be treated is about 20 years of age. In another embodiment, the subject to be treated is about 21 years of age. In another embodiment, the subject to be treated is about 22 years of age. In another embodiment, the subject to be treated is about 23 years of age. In another embodiment, the subject to be treated is about 24 years of age. In another embodiment, the subject to be treated is about 25 years of age.WSGR Ref. No. : 64320-709.601In another embodiment, the subject to be treated is about 26 years of age. In another embodiment, the subject to be treated is about 27 years of age. In another embodiment, the subject to be treated is about 28 years of age. In another embodiment, the subject to be treated is about 29 years of age. In another embodiment, the subject to be treated is about 30+ years of age. In any of such embodiments, the subject can be biologically male. In any of such embodiments, the subject can be biologically female. A subject to be treated with the methods described herein can exhibit an improved quality of life or an improved length of life following treatment. Alternatively, or in addition, a subject to be treated with the methods described herein can exhibit an improved quantity of life following treatment. Administration can be via any suitable method including, but not limited to, intravenous infusion, subcutaneous injection, or a parenteral injection.

[0104] The nMPCs, secretomes, and / or exosomes as provided herein are useful for treating ALS. Treatment includes, but is not limited to, partial or complete treatment.

[0105] In one aspect, provided herein is a method of treating ALS in a subject in need thereof, comprising administering to the subject neonatal cardiac mesenchymal progenitor cells, whereby ALS is treated. In one embodiment, nMPCs to be administered in the described methods are human. In one embodiment, nMPCs to be administered in the described methods are allogeneic. In one embodiment, the nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4; and whereby ALS is treated. In another embodiment, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, or SOX2. In another embodiment, the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2. In another embodiment, the nMPCs are further negative for protein expression of p!6INK4aup to at least passage 8. In another embodiment, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, or SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, or p!6INK4a. In another embodiment, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and p!6INK4a. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. In another embodiment, the nMPCs are genetically engineered to overexpress HGF, SCF, SDF - la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1. In another embodiment, the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and p!6INK4a, and (c) genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1.

[0106] Alternatively, or in addition, in another aspect, provided herein is a method of treating ALS in a subject in need thereof, comprising administering to the subject an nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof, whereby ALS is treated. Alternatively, or in addition, inWSGR Ref. No.: 64320-709.601 another aspect, the subject is administered an nMPC secretome (nMPC conditioned media) described herein. In another aspect, the subject is administered nMPC exosomes. In yet another aspect, the subject is administered an nMPC total secretome comprising both an nMPC secretome and nMPC exosomes. In one embodiment, the nMPC secretome and / or nMPC exosomes are (a) positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, HSF1, and / or Akt, and (b) positive for miRNA expression of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, and / or miR-374b- 5p. In one embodiment, the nMPC secretome and / or nMPC exosomes are (a) positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, HSF1, or Akt, and (b) positive for miRNA expression of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, or miR-374b-5p. In another embodiment, the nMPC secretome and / or nMPC exosomes are (a) positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, HSF1, and Akt, and (b) positive for miRNA expression of miR-31-3p, miR-31-5p, miR-3613-5p, miR-582-3p, miR-7641, and miR-374b-5p. In certain embodiments, the nMPC secretome and / or nMPC exosomes are positive for miRNA expression of one, two, three, four, five, or six of miR-31-3p, miR-31-5p, miR-3613-5p, miR- 582-3p, miR-7641, or miR-374b-5p. In some embodiments, the nMPC secretome and / or nMPC exosomes comprise HSF1 at levels higher than HSF1 in the secretome and / or exosomes of aMPCs. Alternatively, or in addition, in another embodiment, an nMPC secretome is positive for one, two, three, four, five, six, seven, eight, nine, ten, or more of FUS, TAF15, CAP2, SNX9, PSMB7, SUM01, EIF3A, ECHI, HNRNPL, NTM, ANG, ERP44, NAA38, RRM2B, RAB1A, RAB1B, PGRMC1, ERH, TIGAR. ITGA2, SGTA, AR1C1, AKR1C2, DNASE2, HSP90AB4P, ANGPTL2, SF3B2, GREM1, ARF3, ARF1, ARF5, LSM5, PSMD9, SUGT1, ALDH1A3, PDGFRB, HIST1H1C, SP100, G6PD, PPIL1, SNRPD1, FDPS, RAB6B, RAB6A, DCTD, GLB1, ARIH1, IST1, HNRNPU, ULBP2, RAET1G, ERAP2, CDC42, EDF1, LIPG, FKBP4, RBMX, RBMXL1, RPL30, THY1, AP2M1, RPS16, FHL3, BZW1, BZW2, EIF4G1, PFDN4, CLTB, PIR, MVP, ECU, DCI, ALYREF, RPS10, PPP3CA, TAX1BP3, PPA1, DDX39B, DDX39A, GNPNAT1, LIMA1, SNRPD3, ADH5, ENAH, DYNLL1, DYNLL2, HUWE1, HMGB2, PTMS, UCHL3, PFDN2, DYNLRB1, HDGFRP2, INF2, HPRT1, EIF2S2, ADAMT7, EIF3C, EIF3CL, RPS5, VPS35, CS, ECHS1, UBQLN1, GBP2, GBP1, SF3B1, RANBP1, GNS, HLA-B, UBXN1, GNPDA1, IGF2, EIF3B, TCEB1, VASP, GSR, HLA-A, HLA-HIGF2, NUDT5, COPE, HNRNPA3, SRI, HNRNPC, MMP10, EWSR1, IPO7, YARS, TMPO, HNRNPH1, HLA-A, PDGFC, IL1B, S100A10, TXNDC2, DPY30, LAMP1, LYPLA1, CBR1, ARHGAP1, TPR, NAMPT, NAMPTL, CXCL2, EIF4A2, RPS4X, PABPN1, PHLDB1, NAP1L4, DDX6, PSMC3, MAP7D1, OSCAR, TES, NMT1, NMT2, MAT2A, PRMT1, C0X17, SELM, CARS, PPP2CA, PPP2CB, VEGFC, VPS26A, PXN, PAWR, STRAP, RABI 1 A, RABI IB, EIF5, COPZ1, COPS3, CHMP4B, PDXK, RALA, RALB, SEC13, FHL2, TOMI, PYCARD, PDLIM4, RBBP4, RBBP7, RPS20, SRSF1, DLD, SNRPE, EIF4EBP1, RPLPO, RPLP0P6, LSM3, SLC16A3, CSE1L, NUCKS1, TSNAX, RPSA, RPSAP58, AP1B1, EIF2S1, LCP1, PSMD4, AARS, SRSF2, SFRS2, TRIP10, CLTA, USO1, VBP1, UBE2K, MRPL12, UAP 1, HIST1H1B, YBX3, RPL10A, IDH1, SNRPN, SNRPB, PCK2, EIF6, MMP3, DHX9, MATR3, UBE2D2, UBE2D3, PTRF, ACTR1A, LAMA1, PDCD6IP, QARS, HN1L, PLBD2, OS9, ILF3, ISOCI, PPP1R7,WSGR Ref. No.: 64320-709.601MAPK1, PDCD1LG2, GOLGB1, HSPA9, ACBD3, BTF3, UBE2I, SRRT, EIF4H, FERMT2, EIF3G, PARVA, DBNL, BOLA2, MAPRE1, PRDX5, STX12, EIF1, SMS, GRPEL1, OAF, SYNCRIP, ECE1, VAPA, PCNP, PSME1, EIF3J, C14orfl66, DDX1, AKR1B1, SRP9, PCMT1, XPO1, MMP9, RPS7, NRCAM, FKBP3, SSB, DSC3, XRCC5, CCT6A, LIF, SERPINA9, SND1, ANP32B, G3BP1, GPC6, GLOD4, RRBP1, TGFB1, USP14, PLAUR, CXCL3, FASN, COPB2, TARDBP, TNFAIP6, RPS3, NDUFAB1, LMNB1, COL16A1, PSMF1, PHGDH, PRKCDBP, CXCL5, YAP1, TROVE2, MACF1, EIF5B, STX7, PFDNS, APOA1, PDAP1, STX12, EIF3K, FH, RBM8A, EIF1, RPS2, PTGR1, EEA1, CAB39, DCTN2, GDF15, MYL9, TCP1, RPS12, RAD23A, SKP1, PDLIM1, HSPH1, PSMB3, SUB1, API5, TWF2, KTN1, DYNC1H1, SNRNP70, MCFD2, PPP1R18, PTGES3, APEX1, TNKS1BP1, FUBP1, VAT1, PAFAH1B1, CBX1, PTK7, HNRPDL, ZNF185, DYNC1I2, KHDRBS1, SFPQ, PDCD5, TRIM28, PPIC, NARS, PCBP2, CD2AP, PSMA1, CAPRIN1, KHSRP, NSFL1C, PPP1R12A, NUDC, MFAP2, IGF2R, RGMB, PABPC1, MAN1A1, DDB1, PTBP1, FBN2, CNBP, OTUB1, NASP, HSPD1, EEF1D, RNPEP, CRKL, HNRNPAB, EIF3F, PAICS, ILF2, COPB1, PA2G4, COP A, NT5E, HMGA1, SF1, XRCC6, GNB2L1, MEI, MTAP, CCT2, ARCN1, PEPD, and EPB41L3. Alternatively, or in addition, an nMPC secretome is positive for one, two, three, four, five, six, seven, eight, nine, ten, or more ofPPPICA, PPP1CC, STI 3. ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8-MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP, NAP1L1, HSP90AA1, SET, EN01, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, 42254, and PAFAH1B2. In one instance, the nMPC secretome does not comprise 42254. Alternatively, or in addition, an nMPC secretome is positive for one, two, three, four, five, six, seven, eight, nine, ten, or more of FUS, TAF15, CAP2, SNX9, PSMB7, SUM01, EIF3A, ECHI, HNRNPL, NTM, ANG, ERP44, NAA38, RRM2B, RAB1A, RAB1B, PGRMC1, ERH, TIGAR, ITGA2, SGTA, AR1C1, AKR1C2, DNASE2, HSP90AB4P, ANGPTL2, SF3B2, GREM1, ARF3, ARF1, ARF5, LSM5, PSMD9, SUGT1, ALDH1A3, PDGFRB, HIST1H1C, SP1OO, G6PD, PPIL1, SNRPD1, FDPS, RAB6B, RAB6A, DCTD, GLB1, ARIH1, IST1, HNRNPU, ULBP2, RAET1G, ERAP2, CDC42, EDF1, LIPG, FKBP4, RBMX, RBMXL1, RPL30, THY1, AP2M1, RPS16, FHL3, BZW1, BZW2, EIF4G1, PFDN4, CLTB, PIR, MVP, ECU, DCI, ALYREF, RPS10, PPP3CA, TAX1BP3, PPA1, DDX39B, DDX39A, GNPNAT1, LIMA1, SNRPD3, ADH5, ENAH, DYNLL1, DYNLL2, HUWE1, HMGB2, PTMS, UCHL3, PFDN2, DYNLRB1, HDGFRP2, INF2, HPRT1, EIF2S2, ADAMT7, EIF3C, EIF3CL, RPS5, VPS35, CS, ECHS1, UBQLN1, GBP2, GBP1, SF3B1, RANBP1, GNS, HLA-B, UBXN1, GNPDA1, IGF2, EIF3B, TCEB1, VASP, GSR, HLA-A, HLA-HIGF2, NUDT5, COPE, HNRNPA3, SRI, HNRNPC, MMP10, EWSR1, IPO7, YARS, TMPO, HNRNPH1, HLA-A, PDGFC, IL1B, S100A10, TXNDC2, DPY30, LAMP1, LYPLA1, CBR1, ARHGAP1, TPR, NAMPT, NAMPTL, CXCL2, EIF4A2, RPS4X, PABPN1, PHLDB1, NAP1L4, DDX6, PSMC3, MAP7D1, OSCAR, TES, NMT1, NMT2, MAT2A, PRMT1, COX17, SELM, CARS,WSGR Ref. No.: 64320-709.601PPP2CA, PPP2CB, VEGFC, VPS26A, PXN, PAWR, STRAP, RABI 1 A, RABI IB, EIF5, C0PZ1, COPS3, CHMP4B, PDXK, RALA, RALB, SEC13, FHL2, TOMI, PYCARD, PDLIM4, RBBP4, RBBP7, RPS20, SRSF1, DLD, SNRPE, EIF4EBP1, RPLPO, RPLP0P6, LSM3, SLC16A3, CSE1L, NUCKS1, TSNAX, RPSA, RPSAP58, AP1B1, EIF2S1, LCP1, PSMD4, AARS, SRSF2, SFRS2, TRIP10, CLTA, USO1, VBP1, UBE2K, MRPL12, UAP1, HIST1H1B, YBX3, RPL10A, IDH1, SNRPN, SNRPB, PCK2, EIF6, MMP3, DHX9, MATR3, UBE2D2, UBE2D3, PTRF, ACTR1A, LAMA1, PDCD6IP, QARS, HN1L, PLBD2, OS9, ILF3, ISOCI, PPP1R7, MAPK1, PDCD1LG2, GOLGB1, HSPA9, ACBD3, BTF3, UBE2I, SRRT, EIF4H, FERMT2, EIF3G, PARVA, DBNL, BOLA2, MAPRE1, PRDX5, STX12, EIF1, SMS, GRPEL1, OAF, SYNCRIP, ECE1, VAPA, PCNP, PSME1, EIF3J, C14orfl66, DDX1, AKR1B1, SRP9, PCMT1, XPO1, MMP9, RPS7, NRCAM, FKBP3, SSB, DSC3, XRCC5, CCT6A, LIF, SERPINA9, SND1, ANP32B, G3BP1, GPC6, GLOD4, RRBP1, TGFB1, USP14, PLAUR, CXCL3, FASN, COPB2, TARDBP, TNFAIP6, RPS3, NDUFAB1, LMNB1, COL16A1, PSMF1, PHGDH, PRKCDBP, CXCL5, YAP1, TROVE2, MACF1, EIF5B, STX7, PFDNS, APOA1, PDAP1, STX12, EIF3K, FH, RBM8A, EIF1, RPS2, PTGR1, EEA1, CAB39, DCTN2, GDF15, MYL9, TCP1, RPS12, RAD23A, SKP1, PDLIM1, HSPH1, PSMB3, SUB1, API5, TWF2, KTN1, DYNC1H1, SNRNP70, MCFD2, PPP1R18, PTGES3, APEX1, TNKS1BP1, FUBP1, VAT1, PAFAH1B1, CBX1, PTK7, HNRPDL, ZNF185, DYNC1I2, KHDRBS1, SFPQ, PDCD5, TRIM28, PPIC, NARS, PCBP2, CD2AP, PSMA1, CAPRIN1, KHSRP, NSFL1C, PPP1R12A, NUDC, MFAP2, IGF2R, RGMB, PABPC1, MANI Al, DDB1, PTBP1, FBN2, CNBP, OTUB1, NASP, HSPD1, EEF1D, RNPEP, CRKL, HNRNPAB, EIF3F, PAICS, ILF2, COPB1, PA2G4, COP A, NT5E, HMGA1, SF1, XRCC6, GNB2L1, MEI, MTAP, CCT2, ARCN1, PEPD, EPB41L3 PPP1CA, PPP1CC, STI 3. ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8-MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP, NAP1L1, HSP90AA1, SET, ENO1, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, 42254, and PAFAH1B2. In one instance, the nMPC secretome does not comprise 42254.

[0107] In some embodiments, the nMPCs are negative for:(i) a mutation in, or modified expression of, a SOD1 gene or RNA;(ii) a mutation in, or modified expression of, chromosome 9, open reading frame 72, optionally wherein the chromosome 9, open reading frame 72 comprises a C9ORF72 gene;(iii) a fused Fus gene mutation;(iv) a mutation in, or modified expression of, a TDP-43 gene or RNA;(v) a mutation in, or modified expression of, a STMN2 gene or RNA;(vi) a mutation in, or modified expression of, a UNC13A gene or RNA;(vii) a mutation in, or modified expression of, a TARDBP gene or RNA;WSGR Ref. No. : 64320-709.601(viii) a mutation in, or modified expression of, a hnRNPAl gene or RNA;(ix) a mutation in, or modified expression of, a hnRNPA2B 1 gene or RNA;(x) a mutation in, or modified expression of, & MA TR3 gene or RNA;(xi) a mutation in, or modified expression of, an ANG gene or RNA;(xii) a mutation in, or modified expression of, a TUBA4A gene or RNA;(xiii) a mutation in, or modified expression of, an ANXA11 gene or RNA;(xiv) a mutation in, or modified expression of, a PRPH gene or RNA;(xv) a mutation in, or modified expression of, a DCTN1 gene or RNA;(xvi) a mutation in, or modified expression of, a PFN1 gene or RNA;(xvii) a mutation in, or modified expression of, a KIF5a gene or RNA;(xviii) a mutation in, or modified expression of, a UBQLN2 gene or RNA;(xix) a mutation in, or modified expression of, a SQSTM1 gene or RNA;(xx) a mutation in, or modified expression of, an OPTN gene or RNA;(xxi) a mutation in, or modified expression of, a VCP gene or RNA;(xxii) a mutation in, or modified expression of, a CHMP2B gene or RNA;(xxiii) a mutation in, or modified expression of, a VAPBIVAMP gene or RNA; or(xxiv) a mutation in, or modified expression of, a TBK1 gene or RNA;(xxv) a mutation in, or modified expression of, an EphA4 gene or RNA;(xxvi) a mutation in, or modified expression of, a NEK-1 gene or RNA; or (xxvii) a mutation in, or modified expression of, an ATXN2 gene or RNA.

[0108] In some embodiments, the subject is administered one or more IV bags comprising the nMPCs. In some embodiments, the subject is administered more than one IV bag, and each bag is administered at about every 28 days.

[0109] Alternatively, or in addition, in another aspect, the subject is administered nMPC exosomes described herein.

[0110] In any of such methods, a symptom of ALS is improved by 2% or more, or by 2-fold or more, following treatment as compared to a placebo or compared to the subject prior to treatment. In some cases, a symptom is decreased about 1. 1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10- fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1. 1 to about 5 -fold, about 1. 1 to about 6- fold, about 1. 1 to about 7-fold, about 1. 1 to about 8-fold, about 1. 1 to about 9-fold, about 2 to about 5- fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5- fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to a subject treated with a placebo or compared to the subject prior to treatment. In some embodiments, a symptom is improved about 1. 1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1. 1 to about 5 -fold, about 1. 1 to about 6-fold, about 1. 1 to about 7-fold, about 1. 1 toWSGR Ref. No. : 64320-709.601 about 8-fold, about 1. 1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to a subject treated with a placebo or compared to the subject prior to treatment.

[0111] In some embodiments, a symptom of ALS improves by about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to after treatment with a placebo and as compared to a symptom of the disease or condition prior to treatment with any composition described herein.

[0112] In certain embodiments, the symptom of ALS comprises high levels of pro-inflammatory cytokines (e.g., Interleukin (IL)- 1(3, IL-6, IL-8, IL-12, IL-17a, IL-18, MCP-1, tumor necrosis factor (TNF)-a, interferon gamma (IFN-y)). In some embodiments, the treatment reduces levels of pro- inflammatory cytokines by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof such that the level of pro- inflammatory cytokines is considered clinically normal. In an embodiment, the treatment reduces levels of IL- 1 P by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In one embodiment, the treatment reduces levels of IL-6 by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In another embodiment, the treatment reduces levels of IL-8 by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In yet another embodiment, the treatment reduces levels of IL- 12 by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In certain other embodiments, the treatment reduces levels of IL- 17a by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In another embodiment, the treatment reduces levels of IL- 18 by at least aboutWSGR Ref. No. : 64320-709.6012%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In one embodiment, the treatment reduces levels of MCP-1 by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In another embodiment, the treatment reduces levels of INF -a by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In some embodiments, the treatment reduces levels of IFN-y by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In another embodiment, the treatment reduces levels of TNF-a, IL-8, MCP-1, IL- 173, and IFN-y by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In another embodiment, the treatment reduces levels of TNF-a, IL-8, and MCP-1 by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of the nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In another embodiment, the treatment reduces levels of IL-17a and IFN-y by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of the nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof.

[0113] In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is above about 10 pg / ml, about 20 pg ml, about 30 pg / ml, about 40 pg / ml, or about 50 pg / ml prior to treatment. In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is reduced by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream of the subject is reduced by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment. In someWSGR Ref. No.: 64320-709.601 embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the cerebrospinal fluid (CSF) ofthe subject is reduced by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is reduced by at least about 1 pg / ml, 2 pg / ml, 3 pg / ml, 4 pg / ml, 5 pg / ml, 6 pg / ml, 7 pg / ml, 8 pg / ml, 9 pg / ml, 10 pg / ml, about 20 pg ml, about 30 pg / ml, about 40 pg / ml, or about 50 pg / ml compared to treatment with a placebo or compared to the subject prior to treatment.

[0114] In some embodiments, a level of interleukin 8 (IL-8), lipopolysaccharide binding protein (LBP), cluster of differentiation 14 (CD14), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), Insulin-like Growth Factor 1 (IGF-1), Epidermal Growth Factor (EGF), Transforming Growth Factor Beta 1 (TGF- 1), TGF- 2, TGF- 3, neurotrophin-3 (NTF-3), hepatocyte growth factor (HGF), growth differentiation factor 15 (GDF-15), heparin-binding epidermal growth factor-like growth factor (HB-EGF), chitinase-3-like protein 1 (CHI3L1) (Gp39), connexin 39 (Cx39), Cyclophilin B (CypB) (PPIB), TATA-Binding Protein (TBP), DNA-directed RNA polymerase II subunit RPB1 (POLR2A), Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), beta actin (ACTB), or a combination thereof of the subject is modulated by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of IL-8, LBP, CD14, BDNF, GDNF, IGF-1, TGF-J31, TGF- 2, TGF- 3, GDF-15, HB-EGF, CHI3L1 (Gp39), Cx39, PPIB, ACTB, or a combination thereof of the subject is reduced by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of EGF, PPIB, GAPDH, or a combination thereof of the subject is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to treatment with a placebo or compared to the subj ect prior to treatment. In some embodiments, alevel of interleukin ip (IL- ip), IL-6, IL-10, TNF-a, or a combination thereof of the subject is modulated by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Phosphorylated Neurofilament Heavy Chain (pNfH), TDP-43, FUS, SOD1, a dipeptide repeat (DPR) protein, tau protein, phosphorylated tau protein (p-tau), clusterin, a chitinase-like protein, creatine kinase (CK), Glial Fibrillary Acidic Protein (GFAP), or a combination thereof of the subject is modulated by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to treatment with a placebo or compared to the subject prior to treatment. In some embodiments, a level of Phosphorylated Neurofilament Heavy Chain (pNfH), TDP-43, FUS, SOD1, a dipeptide repeat (DPR)WSGR Ref. No. : 64320-709.601 protein, tau protein, phosphorylated tan protein (p-tau), clusterin, a chitinase-like protein, creatine kinase (CK), Glial Fibrillary Acidic Protein (GFAP), or a combination thereof of the subject is reduced by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%compared to treatment with a placebo or compared to the subject prior to treatment.

[0115] In some embodiments, the treatment reduces oxidative stress in a subject by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In some embodiments, the treatment improves motor neuron condition in a subject by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In some embodiments, the treatment improves motor function or control in a subject by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In some embodiments, the treatment reduces muscle twitching or cramping in a subject by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof. In certain embodiments, administration of a composition described herein inhibits or reduces motor neuron loss in a subject by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% as compared to a placebo or compared to the subject prior to administration of nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof.

[0116] In any of such embodiments, the subject to be treated can be a mammal, wherein the mammal is a human. In some embodiments, the human is a child of from birth to 18 years of age. In some embodiments, the human is a child at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age. In some embodiments, the human is an adult of over 18 years of age. In some embodiments, the human is an adult over about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 years of age. In other embodiments, the subject is ambulatory. In yet other embodiments, the subject is non-ambulatory.

[0117] In one aspect, compositions of the present disclosure are administered via intracranial, intracerebroventricular (ICV), intrathecal (IT), or intravenous (IV) infusion. In some embodiments,WSGR Ref. No. : 64320-709.601 compositions of the present disclosure are administered via intravenous infusion. In some embodiments, compositions of the present disclosure are administered via intracranial infusion. In some embodiments, compositions of the present disclosure are administered via intracerebroventricular infusion. In some instances, a first dose of nMPCs is delivered by intravenous infusion over the course of about 10, 15, 30, 45, 60, or 75 minutes. In one non-limiting example, a dose of nMPCs is delivered by intravenous infusion over the course of from about 30 minutes to about 90 minutes (e.g, 60 minutes). In another nonlimiting example, a dose of nMPCs is delivered by intravenous infusion over the course of from about 30 minutes to about 180 minutes (e.g, 90 minutes).Combination Therapies

[0118] In some embodiments, the compositions or formulations comprising the nMPCs, nMPC secretome, nMPC exosomes, nMPC total secretome, or a combination thereof (e.g, secretomes and / or exosomes) disclosed in the present disclosure can be used in combination with an additional therapeutic agent. In some embodiments, the therapeutic agent can comprise a small molecule. In some embodiments, the therapeutic agent can comprise a pharmaceutical drug. In some embodiments, the therapeutic agent can comprise Riluzole, Edaravone, Tofersen, Nuedexta, sodium phenylbutyrate- taurursodiol (PB-TUDCA), or a combination thereof.Animal Models of ALS

[0119] Animal models of amyotrophic lateral sclerosis (ALS) provide a means for studying this incurable and fatal human disease and assessing nMPCs or nMPC secretomes described herein. Postmortem ALS tissue remains the “gold standard” against which pathologic findings in animal models are compared. Four natural disease models have been studied, including mouse models: motor neuron degeneration (Mnd), progressive motor neuronopathy (pmn), wobbler, and one canine model: hereditary canine spinal muscular atrophy (HCSMA). The wobbler mouse has been the most extensively studied of these models with analyses of clinical, pathological (perikaryon, axon, muscle), and biochemical features. Experimentally induced ALS animal models allow for controlled testing of neurotoxic, viral and immune-mediated mechanisms. Mouse models in which genes relevant to the human disease or motor neuron biology can be manipulated have been developed. Transgenic mouse overexpressing the mutated SOD1 gene of FALS patients, provide insights into mechanisms of motor neuron. A transgenic mouse model with mutant TDP-43 appears to be similar to human sporadic amyotrophic lateral sclerosis. Another mouse model is the A315T stop mouse model (see, e.g., Wegorzewska. 2009, PNAS, 106, p. 18809-14 from Jackson Laboratories Inc.). Another exemplary mouse model is the transgenic SOD1 (superoxide dismutase 1) mice (described in the Examples section which follows) which express a G93A mutant form of human SOD1. SOD1 mice (TgN-SODl-G93A-lGur) exhibit a phenotype similar to amyotrophic lateral sclerosis (ALS) in humans (see, e.g., Gurney, 1994, Science 264 p. 1772-5).Certain Definitions

[0120] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is toWSGR Ref. No. : 64320-709.601 be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0121] In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0122] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0123] Groupings of alternative elements or embodiments of the disclosure herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified, thus, fulfilling the written description of all Markush groups used in the appended claims.

[0124] The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g, the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of ± 10% a given value. Alternatively, particularly with respect to bi ological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0125] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited 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 the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.WSGR Ref. No. : 64320-709.601

[0126] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment,” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0127] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub -range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of from 1 to 50 may comprise from 1 to 10, from 1 to 20, from 1 to 30, and from 1 to 40 in one direction, or from 50 to 40, from 50 to 30, from 50 to 20, and from50 to 10 in the other direction.

[0128] The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or anon-human mammal, such as a non-human primate, bovine, equine, canine, ovine, feline, etc.

[0129] The term “optional” or “optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

[0130] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosures.

[0131] The terms “treating,” “treatment,” and the like are used herein to mean obtaining a desired pharmacologic and / or physiologic effect. In some instances, an individual (e.g., an individual suspected to be suffering from and / or genetically pre-disposed to a disease or disorder is treated prophylactically with a preparation of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof as described herein and such prophylactic treatment completely or partially reduces the likelihood of developing a disease or disorder or sign or symptom thereof. In some instances, an individual is treated therapeutically (e.g, when an individual is suffering from a disease or disorder), and such therapeutic treatment causes a partial or complete cure for the disease or disorder and / or reverses an adverse effect attributable to the disease or disorder and / or stabilizes the disease or disorder and / or delays progression of the disease or disorder and / or causes regression of the disease or disorder. In some embodiments, the disease or disorder is a liver-associated, cardiac-associated, immunodeficiency -WSGR Ref. No. : 64320-709.601 associated, nervous system- associated, hemopoietic system-associated, kidney-associated, or cancer- associated disease or disorder.

[0132] Administration (e.g, transplantation) of nMPCs, nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof as disclosed herein to an area in need of treatment is achieved by, for example and not by way of limitation, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as inelastic membranes, or fibers.

[0133] “Transplanting” a composition into a mammal refers to introducing the composition into the body of the mammal by any method established in the art. The composition being introduced is the “transplant,” and the mammal is the “recipient.” The transplant and the recipient can be syngeneic, allogeneic, or xenogeneic. Further, the transplantation can be an autologous transplantation.

[0134] The term “isolated” or “purified” refers to a product, compound, or composition which is separated from at least one other product, compound, or composition with which it is associated in its naturally occurring state, whether in nature or as made synthetically.

[0135] “Non-naturally occurring” as used herein, refers to a compound, product, or composition that is not found in nature (e.g, not endogenous).

[0136] “Allogeneic” as used herein, refers to deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated but genetically similar. An “allogeneic transplant” refers to transfer of cells, a secretome, and / or an exosome from a donor to a recipient, where the recipient is the same species as the donor.

[0137] An “effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose. An effective amount of a composition to treat or ameliorate a disorder is an amount of the composition sufficient to reduce or remove the symptoms of the disorder.

[0138] As used herein, the terms “modulate,” “modulates,” “modulating,” and “modulation” can refer to the upregulation or downregulation of a gene. As used herein, “upregulation” refers to an increase of 0.5% or more compared to a control. As used herein, “downregulation” refers to a decrease of 0.5% or more compared to a control.

[0139] The term “expression” with respect to a gene or polynucleotide refers to transcription of the gene or polynucleotide and, as appropriate, translation of an mRNA transcript to a protein or polypeptide. Thus, as will be clear from the context, the expression of a protein or polypeptide results from transcription and / or translation of the open reading frame.

[0140] As used herein, “negative” refers to a level of expression of about 1.5% or less compared to a control. As used herein, a “low level” refers to a level of expression of about 15% or less than a control. By way of example only, negative can mean the level of detectable transcripts of said gene is 1.5% or less of the total detectable transcripts whereas low can mean the level of detectable transcripts is between 1.6% to about 15% of the total detectable transcripts. Alternatively, negative can mean that a level of expression results in no functional activity of an encoded protein, whereas low can mean a level of expression that results in reduced functional activity as compared to a control level.WSGR Ref. No. : 64320-709.601

[0141] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology, and / or recombinant DNA.

[0142] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0143] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.EXAMPLES

[0144] The application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are presented in order to more fully illustrate embodiments and should in no way be construed, however, as limiting the broad scope of the application.Example 1. Culturing Neonatal Cardiac Mesenchymal Progenitor Cells.

[0145] Described in this Example are the methods of manufacture of novel neonatal cardiac mesenchymal progenitor cells (nMPCs), nMPC secretome, and / or nMPC exosomes and their methods of isolation and culturing. Neonatal cardiac mesenchymal progenitor cells (nMPCs) are a somatic cellular therapy product. Allogeneic nMPCs are derived from fresh human cardiac tissue prepared from donors aged <30 days old using manufacturing processes described herein that enhance their therapeutic benefit and confer properties different from the properties of the cells in their natural tissue environment. For clinical applications, human neonatal heart derived stromal cells are expanded ex vivo to a homogenous cell population with defined cell surface molecules. The final product, comprising neonatal cardiac mesenchymal progenitor cells (nMPCs), nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof, can confer therapeutic functions through its unique and highly potent secretome which has immunomodulatory and anti-apoptotic effects, and aids the restoration of recipient tissue function. A master cell bank comprising nMPCs are cryopreserved at passage 4. Neonatal cardiac mesenchymal progenitor cells (nMPCs), nMPC secretomes, nMPC exosomes, nMPC total secretome, or a combination thereof can be administered to patients in need thereof to treat a symptom of a disease or condition, wherein the disease or condition comprises a wound. The nMPCs, nMPC secretomes, nMPC exosomes, and nMPC total secretome exert therapeutic benefits such as inducing regeneration, reducing progression of neurodegeneration, reducing inflammation, reducing oxidative stress, and offsetting the pathophysiological consequences of a wound by recruiting regenerative factors and reducing systemic inflammation. In certain embodiments, nMPCs are (a) positive for cell surface protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4. The nMPCs can be further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, or SOX2. The nMPCs can be further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2. The nMPCs can be further negative for protein expression of pl6INK4aup to at least passage 8. TheWSGR Ref. No. : 64320-709.601 nMPCs can be (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, or SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, or pl6INK4aThe nMPCs can be (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a. In some embodiments, nMPCs express HSF1 at levels higher than aMPCs. The nMPCs can be genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1. The nMPCs can be (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and p!6INK4a, and (c) genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1.

[0146] The isolation of the nMPCs is performed using a novel protocol in which c-Kit+(CD117+) cells are isolated using specific c-Kit+magnetic beads (as an example of a cell-isolating substrate) and cultured in embryonic progenitor cell medium, as described in this Example.Right Atrial Appendage Collection

[0147] Source tissue for the production and culturing of neonatal cardiac mesenchymal progenitor cells as described herein are selected based on two categories of inclusion: characteristics of the neonates and characteristics of their mothers. First, neonates are of an age less than or equal to 30 days old at the time of surgery, already require neonatal cardiac surgery with cardiopulmonary bypass for congenital heart disease and have no previously known chromosomal abnormalities determined by microarray analysis on the fetal amniotic fluid or post-delivery on the cord blood. Second, all mothers of the neonates must be the biological mother of the neonate, must test negative on viral tests, and are screened for transmissible infectious diseases with assays comprising Hepatitis B surface antigen (HBsAg); Anti -Hepatitis B core antibody (HBcAb); Anti -Hepatitis C virus antibody (HCV Ab); Anti-Human Immunodeficiency Virus (HIV) antibody (HIV 1 / 2); Cytomegalovirus antibody (CMV); HCV / HIV Nucleic Acid test; West Nile Virus Nucleic Acid test; Rapid Plasma Reagin (RPR); Human T-lymphotropic Virus I / n (HTLV I / II); T. cruzi ELISA test (Chagas disease); and / or Zika Virus Testing. Furthermore, a neonate may be excluded if the neonate’s biological mother has any previously known, pathological chromosomal abnormality, has a positive viral test result for a transmissible infectious disease, is unwilling to participate in the study, or is unwilling or not able to provide assent for the neonate.

[0148] The right atrial appendage (RAA) is harvested from neonates under sterile, aseptic conditions before preparation and transfer for additional cell processing. Collection happens in two phases, comprising the preparation of a collection kit and subsequent harvesting. First, a collection kit is prepared by filling each of two 2-ml cryogenic storage vials with about 1.5 ml of HTS-FRS solution. One cryogenic storage vial is prepared to receive the tissue sample, and the second vial is prepared as a backup. A 15-ml centrifuge tube and a sterile plastic bag are also included in the collection kit. After all vials and the centrifuge tube are labeled with a patient ID number, both vials, the centrifuge tube, and theWSGR Ref. No. : 64320-709.601 sterile plastic bag are placed in a biological safety cabinet (BSC). The 15-ml centrifuge tube can only be opened at the harvest site. After each 2-ml cryogenic storage vial is filled with 1.5 ml of HTS-FRS solution, each vial’s cap is screwed tightly, inserted into the sterile bag, and placed in a NANOCOOL™ Cold Chain Shipping container or other validated cooler at 2-8 °C comprising a temperature data logger. This complete collection kit is then transported to a Harvest Site.

[0149] At the Harvest Site, the box comprising the above-prepared tissue collection kit is opened in a sterile environment. The data comprising temperature from the temperature logger are transferred.Surgically collected right atrial appendage tissue is placed into one sterile cryo vial comprising the 1.5 ml of HTS-FRS solution, placed in the sterile 15-ml centrifuge tube, inserted into the sterile plastic bag, and placed into another NANOCOOL™ Cold Chain Shipping container with the activated temperature data logger. After the cryogenic storage vials comprising the RAA tissue are placed in the NANOCOOL™ Cold Chain Shipping container and the temperature data logger is activated, the NANOCOOL™ Cold Chain Shipping container is activated and transported back to the cell manufacturing laboratory with overnight shipping. The cryogenic storage vial is labeled with information comprising the study ID number, age of the neonate, sex of the neonate, date and time of collection, and neonate’s diagnosis.

[0150] As used herein, “passage” or “passaging” refers to the process of culturing, expanding, and harvesting the nMPCs, typically represented as a number.Donor Qualification

[0151] All appropriate Donor Qualification tests are confirmed to have been performed, in accordance with Donor Eligibility Determination Procedure prior to the start of processing.Enzymatic Digestion of Heart Tissue

[0152] All necessary supplies to collect sterility samples and those required for processing heart tissue in the Biological Safety Cabinet are assembled. Collagenase Stock Solution (CSS) and Collagenase Working Solution (CWS) are prepared. Complete Medium comprising Rooster Basal™-MSC-CC (Rooster Bio Cat # SU-022) and Rooster Booster™-MSC-CC (Rooster Bio Cat # SU-019) is also prepared. In some instances, the Complete Medium comprises about 500 ml of Rooster Basal ™-MSC- CC (Rooster Bio Cat # SU-022) and about 10 ml Rooster Booster ™-MSC-CC (Rooster Bio Cat # SU- 019). RAA tissue sample in the cryogenic storage vial is removed from the sealable plastic bag and wiped thoroughly with 70% alcohol or equivalent disinfectant saturated wipe. Label information is verified at this time. The 1 -ml tube containing the heart tissue is aseptically placed in the BSC. One ml of transport media is sterilely removed from the cryovial while care is taken not to aspirate heart tissue from the tube. The RAA tissue biopsy is aseptically transferred into a 100-mm cell culture plate containing 1 ml of Fresh F- 12 Nutrient Mixture (HAM) and weighed. Heart tissue from the culture plate containing F - 12 Nutrient Mixture (HAM) is removed and placed into a 15-ml tube containing 5 ml of 320 U / ml collagenase solution. For instances in which the heart tissue is larger than the tip of a pipette, the tissue is minced using sterile scalpel. The RAA tissue is incubated at 37° C for 20 min. After the incubation, the tissue solution is pipetted up and down. Tissue is allowed to settle at the bottom of the tube for approximately 1 minute or until the tissue is visibly settled down completely. Supernatant is removed andWSGR Ref. No. : 64320-709.601 transferred into anew 15-ml conical. Five ml of Complete Medium is added to neutralize the enzymatic action and the mixture is centrifuged at 500 * g for 5 min at 15 °C. The pellet is resuspended in 5 ml of Complete Medium. The cell suspension is kept cold until all the fractions are collected. To have the optimal digestion, the tissue left in the first tube is exposed to the enzyme two more times by repeating the protocol at the collagenase incubation step.

[0153] The three supernatant fractions collected during the digestion of heart are combined and centrifuged at 500 * g for 5 min at 15 °C. The cells are resuspended in 5-ml Complete Medium and plated in one or two T25 flasks depending on the amount of tissue processed. The cells are incubated at 37 °C, 5% CO2. Heart tissue is also resuspended in Complete Media and plated in one or two T25 flasks depending on the amount of tissue processed, followed by incubation at 37 °C, 5% CO2. The cells are split when half of the number of flasks reaches greater than about 70% and less than about 95% confluence.Passage 0 Feeding

[0154] All necessary supplies required for feeding of cell culture flasks are assembled in the BSC. Cultures are inspected for any signs of contamination before each feeding. Complete Media is prepared. After 72 hours, non-adherent cells are removed by aspiration and fresh Complete Media is added to the adherent cells. Complete Media is replaced every 72 - 96 hours until cells reach about 70-95% confluency. If tissue is not attached to the surface of flask within 3 days of initial plating, only a small amount of new Complete Media is added to the flask as replenishment, but media already present is not completely replaced. For heart tissue, cell sprouting from the edges of the tissue is observed under a microscope. Culture flasks are transferred aseptically to the BSC. Media is manually and gently aspirated media from each flask. Fresh Complete Media is added to the flask with the digested heart tissue. Place them back in the incubator with 5% CO2 at 37°C. When cells from heart tissue and supernatant are about 70-95% confluent, cells are harvested by selecting for those that are positive for cell surface protein expression of CD117 using magnetic selection.Passage 0 Harvest

[0155] The confluence (cell density) of each flask is verified before trypsinizing. Media is gently aspirated from each flask. TrypLE is added. Flasks are observed for cell and heart tissue detachment. If all are detached from the flask(s), TrypLE activity is neutralized by adding equal to or more than the amount of Complete Media. If there are many cells that remain attached, the flasks are gently tapped to lift the adherent cells before adding media to neutralize trypsin. Cells and heart tissue are transferred from each respective flask into two separate, sterile, 15-ml conical tubes. Complete Media is added to each flask and swirled to remove any remaining cells. The remaining media is pooled, and each conical tube is centrifuged at 500 * g for 5 minutes at 15 °C temperature. The supernatant is aspirated, and the cells resuspended in 5 ml of Complete Media. The cell suspensions are pooled into one tube and counted and tested for viability using Trypan Blue Exclusion. MACS separator is assembled in the BSC, along with collection tube and its rack. For cell sorting, an appropriate column and separator is chosenWSGR Ref. No.: 64320-709.601 according to the number of cells. The column is placed in the magnetic field of a MACS separator. Miltenyi buffer is prepared.Magnetic Selection

[0156] The cell suspension is centrifuged at 500 * g for 5 minutes at 15 °C temperature. The cell pellet is resuspended with 300 pl of cold Miltenyi buffer per IxlO8total cells according to the following guidelines: up to IxlO8total cells: 300 pl; from IxlO8to 2xl08total cells: 600 pl; from 2xl08to 3xl08total cells: 900 pl.

[0157] 100 pl of FcR Blocking Reagent is added per IxlO8total cells according to the following guidelines: up to IxlO8total cells: 100 pl; from IxlO8to 2xl08total cells: 200 pl; from 2xl08to 3xl08total cells: 300 pl.

[0158] 100 pl of CD117 Microbeads are added per IxlO8total cells according to the following guidelines: up to IxlO8total cells: 100 pl; from IxlO8to 2xl08total cells: 200 pl; from 2xl08to 3xl08total cells: 300 pl.

[0159] These are mixed well and incubated for 15 min at 4 °C. Cells are washed 2 times by adding 2 ml of cold Miltenyi buffer. The mixtures are centrifuged at 300xg for 10 min at 4 °C and break set at 5. Supernatant is decanted or aspirated completely.

[0160] The cell pellet is resuspended in 500 pl of Miltenyi buffer per 108cells according to the following guidelines: up to IxlO8total cells: 500 pl; from IxlO8to 2xl08total cells: 1000 pl; from 2xl08to 3xl08total cells: 1500 pl.

[0161] The cell suspension is applied onto the column and flow-through containing unlabeled cells collected. The cell suspension in Miltenyi buffer is added only when the fluid is in the interface between the first and second rings of the cup of the column. The column is washed three times with the appropriate amount of Miltenyi buffer as mentioned above. The column is removed from separator and placed on the appropriate new collection tube. An appropriate amount of Miltenyi buffer is pipetted onto the column to flush out the magnetically labeled cells in a 5 ml conical tube by firmly pushing the plunger into the column.

[0162] Complete Media is added in the same 5 ml conical tube and centrifuged at 500 x g for 5 min at 4 °C, break set at 5. Supernatant is aspirated and the cells are resuspended in 1 ml of Complete Media. Cells are collected for counting viability and cell count. Cells are seeded at a density of 10,000-15,000 cells / cm2and Complete Media added. The cell cultures are placed in the incubator with 5% CO2 and 37 °C.Harvesting Passage 1 and Passage 2 Cell Cultures

[0163] All necessary supplies required for feeding of cell culture flasks are assembled in the BSC. Cultures are inspected for any signs of contamination before each feeding. Complete Media is prepared. The confluence of each flask (70-95%) is verified before adding TrypLE. Media is gently aspirated from each flask and TrypLE added. Incubation with TrypLE Select occurs in the BSC at room temperature. Vessels are inspected after 2 minutes. If cells have not detached from the culture surface, the sides of the vessel are tapped and checked again in 2 minutes. After all are detached from the flask, TrypLE activityWSGR Ref. No. : 64320-709.601 is neutralized by Complete Media. The cell suspension is transferred to the appropriate size conical tubes and centrifuge each conical tube at 500 * g for 5 min at 15°C. The supernatant is aspirated, and cells resuspended in 2-5 ml of Complete Media. Cells are collected for counting viability and count, then seeded at a density of 10, 000-15, 000 / cm2. Cells are harvested when they reach 70-95% confluence.Final Harvest for Master Cell Bank at Passage 3

[0164] Complete Media is allowed to warm up to approximately room temperature prior to use. All work surfaces and the BSC are properly cleaned. All supplies needed for harvest are prepared and all sterile bottles, conical tubes and cryovials labeled. Culture vessels are checked under the microscope and confluency noted. A 50-ml aliquot of spent medium is removed from each vessel and pooled in an appropriately sized container for lab testing. A 30-ml aliquot is transferred into each of 4 x 50 ml conical tubes and 8 ml into 10 x 15 ml conical tubes and set aside until cell count is performed.

[0165] Media is gently aspirated from each flask and TrypLE added. Incubation with TrypLE Select occurs in the BSC at room temperature. Check vessels after 2 minutes and gently tap the sides of the vessel if cells have not detached from the culture surface check again in 2 minutes. Vessels are inspected after 2 minutes. If cells have not detached from the culture surface, the sides of the vessel are tapped and checked again in 2 minutes. After all are detached from the flask, TrypLE activity is neutralized by Complete Media. The cell suspension is transferred to the appropriate size conical tubes and centrifuge each conical tube at 500 * g for 5 min at 15 °C. The remaining supernatant is aspirated without disturbing the pellet and the pellet resuspended in 5-10 ml of CryoStor CSB, the freezing medium. The cells are collected for counting viability and count.Cryopreservation

[0166] The amount of CryoStor CS10 final re-suspension volume needed to obtain a concentration of 2.5xl06cells / ml is calculated based on the following equation:Viable Cells Total 1.25%l 06cells / mL - Cell suspension (mL) = Volume of CS10 mL

[0167] The pellet is resuspended in CryoStor CS 10 and a 2-ml aliquot of cell suspension transferred into each 2-ml cryovial. Aliquots are cryopreserved in a -80 °C freezer. The following day, the Master Cell Bank vials are transferred to a -196°C liquid nitrogen storage tank for long term storage, in quarantine, until the product is found to be sterile and suitable for clinical use.Quality Control of the Master Cell Bank

[0168] Quality control inspection of Master Cell Bank vials comprises rapidly thawing one vial of cryopreserved Master Cell Bank in a 37 °C water bath and slowly diluting with 5 ml Complete Media to assess the lot release criteria Cell Count, Viability, and Flow Cytometry. Cell Count and Viability of the Master Cell Bank vial is assessed via manual counting using a Trypan Blue exclusion (SOP LAB001) and are within 2xl06± 20% cells per vial with a viability of > 80%. At least 2xl06cells of diluted product are transferred to a 15-ml conical tube for cellular phenotype via Flow Cytometry assessment. Samples are processed and analyzed. The samples contain <3% CD45+ expression, and >80% CD105+, CD90+, CD44+, and CD117+ expression.WSGR Ref. No. : 64320-709.601

[0169] One cryopreserved STM-01 Master Cell Bank vial is shipped to Charles Rivers on dry ice for analysis using the FDA approved (USP BET <85>) ENDOSAFE® NexGen-PTS equipment and sampled cells are expected to contain <5.0 EU / ml endotoxin. Two cryopreserved nMPC Master Cell Bank vials are shipped to Charles Rivers on dry ice for mycoplasma testing. Samples are assessed according to USP<63> and are expected to test negative for mycoplasma contamination.

[0170] All in-process sterility samples are frozen and shipped to Charles Rivers. Samples are assessed via the compendial Bacterial and Fungal 14-day culture methods (USP <71>) and are expected to negative for Aerobic and Anaerobic microorganism contamination. Final sterility samples are frozen and shipped to Charles Rivers. Samples are assessed via the compendial Bacterial and Fungal 14-day culture methods outlined in USP<71> and are expected to test negative for contamination. Samples also undergo Adventitious Agent testing and Karyotyping.

[0171] Neonatal cardiac mesenchymal progenitor cells secrete exosomes which are characterized by their specific paracrine expression profile as shown by fluorescence-activated cell sorting (FACS) and electron microscopy properties. In one embodiment, exosomes isolated from medium conditioned by neonatal cardiac mesenchymal progenitor cells can induce recovery of neuronal tissue, reduce inflammatory markers, or reduce oxidative stress more strongly than exosomes derived from medium conditioned by adult-derived mesenchymal progenitor cells due to preferential expression of cytokines and mIRs by the neonatal cells versus the adult cells.

[0172] The neonatal cardiac mesenchymal progenitor cells described herein can be used as an “off-the- shelf’ progenitor cell product for allogeneic intravenous infusion, allogeneic transplantation, intracranial infusion, intracerebroventricular (ICV) infusion, or intrathecal (IT) infusion in patients in need of such treatment. Because these cells express mesenchymal progenitor cell markers, they can be immunologically privileged in the manner of immunologically privileged bone marrow-derived mesenchymal cells.

[0173] Reagents used in Example 1: N Collagenase Type II 1035 U / ml; F-12 Nutrient Mixture (HAM) (IX), liquid + L-Glutamine; ROOSTERBOOSTERT-MSC-CC; Human Serum Albumin; Trypan Blue; and MACS Rinsing Solution.Example 2. Features of Neonatal Cardiac Mesenchymal Progenitor Cells

[0174] One can characterize compositions comprising nMPCs described herein according to the criteria presented in Table 1.Table 1. Features of nMPC CompositionsWSGR Ref. No. : 64320-709.601

[0175] In specific embodiments, the Table 2 notes specific markers or factors present or not present in the cells.Table 2. Markers of nMPCs

[0176] As can be seen from Table 3, secretomes of neonate cardiac mesenchymal progenitor cells (nMPCs) in vivo differ from the secretomes produced by neonate cells in vitro,' secretomes of adult cardiac mesenchymal progenitor cells (aMPCs) in vivo differ from secretomes of adult cells produced in vitro,' secretomes of neonate cells in vivo differ from secretomes of adult cells produced in vivo,' secretomes of neonate cells produced in vitro differ from secretomes of adult cells produced in vitro,' secretomes of neonate cells in vivo differ from secretomes of adult cells produced in vitro,' secretomes of neonate cells produced in vitro differ from secretomes of adult cells in vivo,' and each of the secretomes produced by neonate and adult cells in vitro and in vivo differ from Iscove's Modified Dulbecco's Medium (IMDM)-treated (control) cells in vivo. Accordingly, it is understood that the cells and the secretomes differ with respect to protein expression and are distinct cell populations. Moreover, these results demonstrate that the nMPCs produced in vitro by the methods described above are non-naturally occurring.Table 3. Differences Among Secretomes of Neonatal MPCs and Secretomes of Adult MPCs in vitro and in vivoWSGR Ref. No.: 64320-709.601

[0177] Furthermore, unique markers are found in nMPC secretomes when compared to adult MPC secretomes (Table 4) Neonatal cardiac mesenchymal progenitor cells express unique markers italicized in Table 4 that are not found in secretomes of aMPCs, while markers with >2-fold higher expression in secretomes of nMPCs as compared to secretomes of aMPCs are in bold.Table 4. Unique Markers Found in Secretomes of Neonatal MPCs vs. Secretomes of Adult MPCsWSGRRef. No.: 64320-709.601WSGRRef. No.: 64320-709.601WSGR Ref. No. : 64320-709.601Example 3. The nMPC secretome reduces inflammation in LPS-stimulated human microglia in vitro

[0178] Chronic activation of microglia can lead to irreversible Central Nervous System (CNS) damage. Persistent inflammation in the brain affects neuronal plasticity, impairs memory, and is generally considered a typical driver of tissue damage in neurodegenerative disorders. Microglia are a key regulator of inflammatory responses in the central nervous system. Microglia activation is heterogeneous and traditionally categorized as neurotoxic (Ml -phenotype microglia) or neuroprotective (M2-phenotype microglia). Several treatments have been attempted to dampen neuroinflammation by modulating activated pro-inflammatory microglia into anti-inflammatory phenotype in pre-clinical animal models and in clinical patients. Stem cell therapies have been investigated as anti-inflammatory therapy for neurodegenerative diseases, but few satisfactory treatments have been established. Although stem cell- based therapy may reduce neuroinflammation, most stem cell-based therapies have had mixed or inconsistent results in patients with neurodegenerative diseases in clinical settings.

[0179] Lipopolysaccharide (LPS) is a molecule found on the surface of Gram-negative bacteria that triggers an inflammatory response in cells, and thus, LPS-stimulated microglia are used as an in vitro model to study the mechanisms of neuroinflammation in the brain. In this Example, the antiinflammatory efficacy of human nMPC secretome was evaluated in LPS-stimulated human inflammatory microglia in vitro. Pro-inflammatory cytokines were measured using the MESO SCALE DISCOVERY® diagnostic platform (MSD U-PLEX). The cytokine analysis using human nMPC culture supernatant (e.g, nMPC secretomes) revealed that nMPC secretome significantly reduced inflammatory cytokines such as, for example, TNF-a, IL-8, and MCP-1, compared to untreated LPS-stimulated microglia.

[0180] The microglial cells were divided into four experimental groups: 1) Control group, in which cells were incubated in DMEM / F-12; 2) LPS-treated group, in which cells were incubated in DMEM / F-12 and 100 ng / mL lipopolysaccharide (LPS); 3) Treatment group 1, in which cells were incubated with nMPC secretomes (1 pg / mL) and 1 mg / mL lipopolysaccharide (LPS); and 4) Treatment group 2, in which cells were incubated with nMPC secretomes (10 pg / mL) and 100 ng / mL lipopolysaccharide (LPS). The microglial cells stimulated with LPS served as positive control.

[0181] Randomization: From the collection cell bank, five nMPCs lines were randomly chosen from male patients (n=24) having normal functional myocardium (collected during operations for structural abnormalities), and secretome was collected from these nMPCs lines for all experimental studies.Table 5. Description of Test Article: nMPC secretomesWSGR Ref. No. : 64320-709.601Table 6. Description of Test Article: LPSAnti-inflammatory effect of nMPC secretomes in LPS-stimulated microglia

[0182] The anti-inflammatory properties of nMPC secretomes were assessed using human iPSC-derived microglia (bit. bio #iol029S). The female donor-derived microglia matured upon revival in the recommended media as per the manufacturer protocol. Briefly, the generation of these cells was divided into a four-phase process: (1) an Induction phase carried out at bit. bio; (2) Phase 1: Stabilization for 24 hours; (3) Phase 2: Maturation for a further 9 days; and (4) Phase 3: the Maintenance phase. Cells were ready to use from day 10. Briefly, on day 10, 2 mL of microglial cells were plated into 6-well plates at a 1.5xl05cells / mL density. These cells were divided into four treatment groups and cultured under the abovementioned conditions. After incubating at 37 °C for 24 and 48 hours, the supernatant was collected and centrifuged to remove debris. Levels of pro-inflammatory cytokines including, for example, TNF-a, IL-8, MCP-1, IL-17a and IFN-y, were assessed using the MSD U-PLEX Proinflammatory Kit according to the manufacturer’s instructions.Statistical Methods

[0183] Data were analyzed using GraphPad Prism software. More than two comparisons were made using One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Probability values of less than 0.05 were considered significant and two-sided tests were performed. Data are represented as mean ± SEM. Data are presented as mean and error bars depict the SEM.Materials

[0184] Materials used in this study are listed in Table 7.Table 7. Study MaterialsWSGR Ref. No.: 64320-709.601Equipment

[0185] Equipment used in this study comprise a THERMO FISHER SCIENTIFIC® centrifuge.Results

[0186] Therapeutic efficacy of nMPC secretomes in LPS stimulated microglia in vitro.WSGR Ref. No. : 64320-709.601

[0187] To identify the inhibitory effect of nMPC secretomes, cytokine production by microglia cultured with nMPC secretomes was assessed. Microglia have been shown to release many cytokines, chemokines, and free radicals under inflammatory conditions. After subjecting the cells to the different treatments described in this Example, the supernatants of nMPCs were collected and cytokine levels were analyzed using MSD assays for TNF-a, IL-8, MCP-1, IL-17a and IFN-y. In the presence of LPS, microglia were found to secrete more pro-inflammatory cytokines such as, for example, TNF-a, IL- 8, and MCP-1 than controls; however, nMPC secretomes significantly inhibited secretion of these factors (FIGS. 1A-1E). Inflammatory cytokines including, for example, IL-17a and IFN-y had reduced levels following administration of nMPC secretomes at a concentration of 10 pg / ml in LPS-stimulated microglia, but results were not statistically significant. nMPC secretomes inhibits pro-inflammatory cytokine production by LPS-activated microglia

[0188] Pro-inflammatory cytokine production by microglia was analyzed using the aforementioned MSD assay. LPS-treated microglia produced higher levels of TNF-a (FIG. 1A, bars with triangles), IL-8 (FIG. IB, bars with triangles), and MCP-1 (FIG. 1C, bars with triangles) than control microglia (FIGS. 1A-1C, bars with circles, or no shapes), while treatment with nMPC secretomes (1 or 10 pg / mL) significantly inhibited production of these factors (FIGS. 1A-1C, bars with diamonds for 1 pg / mL. and bars with pentagons for 10 pg / mL). LPS-treated microglia experienced about a 3.8-fold reduction in TNF-a levels relative to the LPS-only condition after treatment with both 1 and 10 pg / mL nMPC secretomes when assessed at 24 and 48 hours (FIG. 1A, columns marked by diamonds and pentagons). LPS-treated microglia experienced about 2-fold reduction in IL-8 levels relative to the LPS-only condition after treatment with 10 pg / mL nMPC secretomes and assessed after 24 hours (FIG. IB, columns marked by pentagons). LPS-treated microglia experienced about an 8-fold reduction in MCP-1 levels relative to the LPS-only condition after treatment with 10 pg / mL nMPC secretomes when assessed at 24 hours (FIG. 1C, fourth column from the left marked by pentagons). Similar to TNF-a, IL-8, and MCP-1, nMPC secretomes (10 pg / mL) also inhibited production of IL-17a (FIG. ID, bars with pentagons) and IFN-y (FIG. IE, bars with pentagons), however, the cytokine reduction was not statistically significant compared to the LPS-treated microglia groups (FIGS. ID- IE, bars with triangles). Data are represented as mean ± SEM. ** p<0.01, ***p<0.001, ***p<0.0001, and one-way ANOVA followed by Tukey’s post hoc test.

[0189] Taken together, these results show that nMPC secretomes modulated LPS-activated microglia with significantly reduced production of the inflammatory cytokines TNF-a, IL-8 and MCP-1 compared to LPS-stimulated microglia not treated with nMPC secretomes.Example 4. Models of Amyotrophic Lateral Sclerosis (ALS)

[0190] The effects of nMPCs and nMPC total secretome (comprising nMPC secretome and nMPC exosomes) on one or more symptoms of ALS are evaluated in SOD1-G93A transgenic mice, which express a G93A mutant form of human SOD1. SOD1 mice exhibit degeneration of spinal motor neurons that results in paralysis of one or more limbs over time and abbreviated life spans. Molecular effects of nMPCs or nMPC secretomes on SOD1 -G93A transgenic mice are examined by measuring the changes inWSGR Ref. No. : 64320-709.601 pro-inflammatory cytokine expression levels and oxidative factor expression levels compared to a placebo treatment group. Physiological effects are assessed in SOD1-G93A transgenic mice by observing changes in their motor ability or imaging of motor neuron samples after treatment with nMPCs or nMPC total secretome. All experiments are conducted with IACUC approval and performed as specified in the license authorized by the national Animal Experiment Board of Finland and according to the National Institutes of Health (Bethesda, MD, USA) guidelines for the care and use of laboratory animals.

[0191] Experimental mice (n=2-20; age ~50 to 60 days old) are infused with nMPCs or nMPC secretome. Control animals are infused with physiological phosphate buffered saline. Mouse life span and neuromuscular function are monitored.

[0192] Animal Identification: Each animal is marked with a unique identification number at the breeding facility (The Jackson Laboratory) using Remnant UID MiniMax microchips. These ear tags are scanned to identify each animal shortly after animal arrival at the Test Laboratory (CRL Finland). Upon assigning animals to housing cages, QR ear tags may be replaced with another tag according to the Test Facility SOPs with a different color in case there would be more than one animal with the same color code in the same cage.

[0193] Environmental Acclimation: After receipt at the Testing Facility, the animals are acclimated for a target of 7 days prior to the start of the in-life phase, i.e., baseline functional testing.Husbandry

[0194] Animal housing: Animals are socially housed with 2-4 mice per cage. Animals of different dosing groups are housed together. Cages are individually ventilated (IVC) NexGen 500 caging systems (Allentown Inc.) Bedding material comprises aspen chips and paper shred. Animal environmental enrichment is provided for the animals according to the Government Decree on the Protection of Animals Used for Scientific or Educational Purposes (564 / 2013). Enrichment in the home cage consists of plastic igloos, gnawing material (aspen sticks), nesting material, and tunnel used for handling. Chow comprises standard chow: S sniff RM Maintenance - Low phytoestrogen, ad libitum. Water comprises tap water, ad libitum. Animals are kept at room temperature (21.5 ± 1.5°C), with a room humidity of 40-70% RH. Light cycle entailed lights on from 7 AM to 8 PM. Pre-Handling: mice are only handled together with normal husbandry routines prior to the start of the in vivo activities in the study. Mice are picked up from their home cage by tunnel- or cup-handling.Veterinary Care and General Health Status

[0195] Veterinary care is available throughout the course of the study. The welfare of the animals is checked at least daily by trained personnel and supervised by a veterinarian when warranted by clinical signs or other welfare-related changes. If animals show signs of illness or distress, recommendations are made on the treatment of the animal(s) and / or alteration of study procedures based on the facility SOPs and study-related guidance, and documented. If the condition of the animal(s) does not improve and the animal(s) reach the pre-defined humane endpoint criteria, the Study Director and Veterinarian act to alleviate the suffering of the animal and perform euthanasia.WSGR Ref. No. : 64320-709.601Humane Endpoint Criteria

[0196] General humane endpoint criteria: An animal that has mild or moderate impairment of general condition (welfare scoring: activity, posture, piloerection, orbital tightening, breathing problems, diarrhea; dehydration, signs of pain, other abnormal behavior, inability to feed, drink, urinate, defecate or groom itself) is tracked and treated according to Veterinary Care SOP for a maximum of 2 days, within what time the animal’s condition should return to normal, otherwise the animal is euthanized. If animal’s general condition is impaired at severe extent, reaching total score of 10 in welfare Scoring, or receiving score 3 in any if the individual aspects of welfare scoring, or receiving score 2 in breathing or diarrhea category (marked dyspnea or profuse diarrhea), it is euthanized immediately. When applying the human end point criteria, the age and genotype of the animals will be considered. More than 5 seconds of delay in righting reflex, body temperature cannot be maintained within 24 hours, hydrocephalus, un solvable malocclusion, severe injury or chronic wound, auto-mutilation, larger than 1 cm tumor in diameter or swelling. Symptoms that are known to be related to the disease progression usually observed in the current disease model are not considered as humane end point criteria.

[0197] Disease model-related humane endpoint criteria: 25% body weight loss compared to maximum body weight of the animal or ALS-score of 1.5. Test substance administration-related welfare complications: if administration of the nMPCs or nMPC secretome causes impairment in the general condition or signs of pain (such as mentioned in “general and model-related humane endpoint criteria”), the dosing is discontinued. If the animal’s condition does not improve (estimation with welfare scoring) for supportive treatment, the animal is euthanized.

[0198] Test and Reference Articles are described in Tables 8A and 8B.Table 8A. Test and Reference ArticlesTable 8B. Test and Reference Articles

[0199] Preparation of Formulations: Dosing formulations are prepared at appropriate concentrations to meet the dose level requirements. The dosing formulations are prepared at a frequency, stored, andWSGR Ref. No. : 64320-709.601 divided into aliquots prior to dispensing as described herein. Any residual volumes from each dosing occasion are discarded.Experimental Design

[0200] Before the initiation of the in-life phase, any animals considered unsuitable for use in the study may be replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. Animals may be excluded, or groups may be switched based on genotype reanalysis. Animals are assigned to the experimental groups using a stratified randomization scheme designed to achieve balanced groups based on body weight and litter. Animals from the same litter are divided between the Test Compound dosing groups and the SOD1 -G93A vehicle group at best effort to minimize the effect of the litter or number of transgene copies on the study outcomes. A schematic of the survival study arm paradigm with nMPC secretome or nMPCs can be found in FIG. 2.Survival study arm with nMPC secretome

[0201] The survival study arm with nMPC secretome comprises animals grouped as follows: Group 1: 25 SOD1-G93A female mice administered with vehicle (intravenous, 7 doses);Group 2: 25 SOD1-G93A female mice administered with nMPC secretome (10 mg / kg, intravenous, 7 doses); andGroup 3: 25 SOD1-G93A female mice administered with nMPC secretome (25 mg / kg, intravenous, 7 doses).Table 9. Summary of treatments for survival study arm with nMPC secretome.* The dose volume may be lowered in case animal weight is 15 g or lower. In that case, dose volume will be 75 pl resulting in a 0. 15 mg or 0.375 mg nMPC secretome dose.Survival study arm with nMPCs

[0202] The survival study arm with nMPCs comprises animals grouped as follows with additional details in Table 10:Group 1: 10 SOD1-G93A female mice administered with vehicle (intravenous, 7 doses);Group 2: 10 SOD1-G93A female mice administered with nMPCs low dose (10 million cells / kg (about 200,000 cells), intravenous, 7 doses); andGroup 3: 10 SOD1-G93A female mice administered with nMPCs high dose (25 million cells / kg (about 400,000 cells), intravenous, 7 doses).WSGR Ref. No.: 64320-709.601Table 10. Summary of treatments for survival study arm with nMPCs.Sampling study arm

[0203] A schematic of the sampling study arm can be found in FIG. 3 and additional details in Table11

[0204] The sampling study arm comprises animals grouped as follows:Group 1: 10 SOD1-G93A female mice administered with vehicle (intravenous, 4 doses);Group 2: 10 SOD1-G93A female mice administered with nMPC (10 million cells / kg (about 200,000 cells) intravenous, 4 doses); andGroup 3: 10 SOD1-G93A female mice administered with nMPC (20 million cells / kg (about 400,000 cells) intravenous, 4 doses).Table 11. Summary of treatments for the sampling study arm with nMPC secretome.Methods

[0205] The in-life procedures, observations, and measurements listed below are performed by personnel blinded to the treatment, unless otherwise stated.

[0206] Survival Study Arm with nMPCs: Cell PreparationsMaterials

[0207] nMPC Vial # XX

[0208] Lot# nMPC-002 XX

[0209] Dose: 20x106cells / 0.2 ml

[0210] Contains: CRYOSTOR® C810

[0211] Store at -15 °CWSGR Ref. No. : 64320-709.601Medium

[0212] 10X Rooster Basal media bottles

[0213] 10X Rooster SupplementProtocol for Cryovial Retrieval and Thawing:

[0214] Cryovials are removed from the liquid nitrogen (LN2) freezer and transported immediately to the cleanroom. The vial(s) are quickly thawed in a 37 °C water bath by gently swirling it. Visual confirmation of residual slush / ice is expected. Disinfection and transfer to laminar flow hood: The vial is wiped thoroughly with 70% isopropyl alcohol (IPA). The vial is cleaned again with an alcohol prep pad in the laminar flow hood.

[0215] Medium Addition and Initial Suspension: The vial is aseptically opened, and 2 ml of pre- warmed Complete Growth Medium (Table 1) slowly added to the vial. The suspension is transferred to a 50-ml conical tube containing 20 ml of pre- warmed medium. Centrifugation: The suspension is centrifuged at 500 x g for 10 minutes at 20° C.

[0216] The supernatant is then inspected; if it is not clear, the centrifugation is repeated.

[0217] Post-centrifugation Handling: The supernatant is carefully removed without disturbing the pellet. After centrifugation step, 1 ml of PBS (lx PBS, pH 7.2 Gibco™ 20012019, without calcium or magnesium) is added to the cell pellet, which is then resuspended slowly to get individual cells and make the volume as per the need for cell count and injection in lx PBS, pH 7.2 Gibco™ 20012019. Cells are collected for viability assessment and cell count using nucleocounter. Doses are prepared (30% extra volume / animal may be prepared if possible) comprising 200,000 cells / 100 pl (Low dose) and IxlO6cells / 100 pl (high dose).

[0218] Cells and vehicle (control) are stored on ice. Dosing instructions: Each time before dosing, the cells are gently mixed with a sterile tipped pipette to minimize the formation of clumps, then injected. The leftover cells and vehicle are discarded after the dosing.

[0219] Complete Medium Preparation can be found in Table 12. No need to filter the media. Fresh medium will be prepared for all thawings and can be used for about two weeks. 500 ml of medium will be prepared for each thawing day.Table 12. Complete Medium PreparationGenotype Confirmation

[0220] Ear samples are collected upon marking the ears of the mice at the pre-treatment start baseline. The samples are used to re-genotype the animals to confirm the presence of the SOD1 -G93A transgene. For ear sample collection, the animals are administered with a single dose of carprofen (5.0 mg / kg, subcutaneous (s.c.)) for analgesia. Animals with a different genotype as compared to the informationWSGR Ref. No.: 64320-709.601 acquired from the vendor are reallocated to another group or excluded and replaced if possible. Genotyping is completed before dosing onset (day 60).

[0221] The assay discriminates hemizygous B6SJL-Tg(SODl*G93A)lGur / J (Jackson Laboratory strain #002726) and WT mice, but does not distinguish copy number variation i.e., high vs. low G93A. The assay (modified from https: / / www.jax. org / Protocol?stockNumber=002726&protocolID=24173) does not distinguish hemizygous from homozygous transgenic mice.

[0222] The collected samples for genotyping will be stored at -80°C or for short time at -20° C (maximum of 4 weeks) until analyzed.

[0223] The DNA in the tissues is released by applying the Phire Tissue Direct PCR Kit (F170S;THERMO SCIENTIFIC®), using the Dilution Buffer and DNA Release Additive according to the manufacturer’s recommendations. Subsequently, 1 pl of the DNA-containing lysate is multiplied in the PCR reaction with internal positive control (WT mouse) primers and transgenic SOD1 -specific primers applying the Phire Tissue Direct PCR Master Mix (THERMO SCIENTIFIC®, Cat. F170S). The products are separated on a 1% agarose gel and visualized with Sybr Safe DNA gel stain (S33102; Invitrogen) using the Gel Doc XR+ Gel Documentation System (Bio-Rad Laboratories, Inc.).

[0224] The expected products are 236 bp (transgenic SOD1) and 324-bp (internal control). Thus, wildtype (WT) mice have one 324-bp band while transgenic (TG) SOD1 -G93A mouse has both a 236-bp and a 324-bp band.Clinical Scoring and Disease Onset

[0225] Clinical scoring is performed once-per-week from arrival until age day 89 at 7-10 AM. On age days 90-115 / 150, scoring is performed thrice-per-week. Body weights are measured co-jointly with clinical scoring. On days with intravenous (i.v.) administration of the Test Compound or vehicle or functional testing, scoring is performed before these activities.

[0226] Disease onset day is registered as the age day on which clinical score 4.0 or lower was assigned for the mouse for the first time.

[0227] Animals are scored by lifting them gently by the base of their tails and observing them for tremors, stiffness, and their ability to extend their limbs.

[0228] The following is a more specific breakdown of the scoring system:

[0229] 5 = Healthy. Very active and usually aggressive. Normal climbing grip and utilizes actively all fingers of all limbs.

[0230] 4 = Climbing grip strength observably weakened: Not fully able to utilize hind limb fingers, does not climb voluntarily and hind limb grip is usually lost quickly. Visible minor tremors when lifting by the tail. All limbs still extend. Very active.

[0231] 3.5 = Minor stiffness of the limbs and minor tremor. Limb splay during gait. Pelvic region still high above the ground. Does not grab the observer’s finger with hind limb upon lifting by the tail. Normal to almost normal activity level.

[0232] 3 = Mild weakness in the limbs. Modestly reduced activity level. Observable stiffness in the limbs. Lowered pelvic area. Modest muscle weakness usually in the foot.WSGR Ref. No. : 64320-709.601

[0233] 2.5 = Modestly reduced activity level. Significant weakness in and labored extension of the limbs. Struggles to walk.

[0234] 2 = Paralysis in hind limbs. Cannot walk and support its weight on hind limbs and is consequently mostly dragging itself with fore limbs. Activity is significantly reduced. Can right itself upon turning on either side in 5 sec.

[0235] 1.5 = Complete paralysis of hind limbs, minor tremor might be observed. Cannot right itself upon turning on either side in 5 sec. Endpoint criterion. The animal is euthanized on the same day.

[0236] 1 = Moribund. Animal is unable to move voluntarily. Animal will be euthanized immediately.Body Weights

[0237] Body weight is measured co-jointly with clinical scoring after assigning a score to the animal at 7-10 AM. Body weights are measured once-per-week from arrival until age day 89 and thrice-per-week on age days 90-115 / 150. The last measurement is performed on day 115 / 150 or earlier at the humane end point.Test Compound or Vehicle Administration

[0238] Test Compound (nMPC secretome) or vehicle is administered to the mice intravenously (i.v., injection volume of 100 pl) on age days 60±l, 74±1, 88±1, 102±l in both study arms, followed by dosing on days 116±1, 130±l and 144±1 in the survival study arm resulting in in total 4 (sampling study arm) or 7 doses (survival study arm) per mouse. Dosing is performed on the following day after Rotarod testing at each of the time points at 8-12 AM after clinical scoring and body weight measurements in case scoring will be performed on the same day. Prior to the first i.v. dose, the mice are habituated to restraining tube. In case the weight of the animal is 15 g or lower, the dose volume is lowered to 75 pl.Grip Strength Test - Survival Arm Only

[0239] The test is performed on days 58 (pre-treatment baseline) followed by testing on days 72±1, 86±1, 100±l, 114±1, 128±1 and 142± 1 duringthe light phase. Testing is performed after clinical scoring and body weight measurement when these activities are scheduled for the same day.

[0240] Prior to the test, all animals are allowed to habituate to the test room for 30 min. The animal will be placed on the measurement platform and allowed to grip the small mesh of the grip strength apparatus (San Diego Instruments, San Diego, USA). The animal is then slowly pulled by its tail until it releases the mesh. The equipment automatically measures the strength of the grip in grams. Five consecutive trials are recorded for each animal and the result is given as the average of the three best scores.

[0241] Neuromuscular function is assessed by neurological score that tests the mouse muscle tonus reflexes and overall in-cage locomotive activity described in Alves C J et al. Brain Res. (2011) Jun. 7; 1394:90-104, incorporated by reference herein. Hang-wire test is utilized; the hang-wire test measures gross physical strength of forelimbs, hindlimbs, capacity to adduct when hand on a pole and to walk. Automated Catwalk gait analysis system is utilized which is based on camera monitored computerized locomotion testing automated system by, for example, Noldus Information Technology, The Netherlands.WSGR Ref. No. : 64320-709.601Rotarod Test

[0242] For the survival arm, the test is performed on days 59 (pre-treatment baseline) followed by testing on days 73±1, 87±1, 101±l, 115±1, 129±1 and 143±1 during the light phase. In the satellite arm, testing is performed on day 114. Testing is performed after clinical scoring and body weight measurement in case these activities are scheduled for the same day.

[0243] Prior to the test, the animals are allowed to habituate to the test room for 60 min. A one-day session includes a training trial of 5 min at 4 RPM on the rotarod apparatus (Med Associates Inc, St Albans, VT, USA). One hour later, the animals are tested for 3 consecutive accelerating trials of 6 min each with the speed changing from 0 to 40 RPM over 360 s. The inter-trial intervals are at least 30 min. The latency to fall from the rod is recorded. The first time that a mouse grabs the rod in such a way that it rotates one full round with the rod without falling, it is recorded as latency to fall. Data is presented as the average latency to fall off the rotarod of three independent rounds per time point and is normalized to average initial performance at pre-symptomatic stage for each mouse for each time point.

[0244] A fully automated gait analysis with the Catwalk XT™ (Noldus) system is performed. Data is obtained of more than 4 independent rounds on the apparatus. For Hangwire muscle strength assessment, mice are allowed forelimb grip onto a 2-mm thick horizontal metal wire, suspended 80 cm above surface. Ability to successfully raise hindlimbs to grip the wire and crawling to the end of the wire is scored: l=Successful four-paw grip and crawling to the end of the wire within less than 60 seconds. 2=Four-paw grip for more than 15 seconds without crawling. 3=Failure to successfully establish hindlimb grip in 60 seconds. 4=Inability to sustain forelimb grip for 60 seconds. Each mouse is evaluated in 2 sessions on 2 consecutive days, consisting of 3 trials each, with a 15 -minute inter-trial interval. The second day is used to obtain the data as displayed.

[0245] Neurological score is assessed according to Gill et al. (2009) PLoS One, 4(8): e6489, incorporated by reference herein: 0 = more than 2 second extension of hindlimbs away from lateral midline when mouse is suspended by its tail 2-3 times. 1 = collapse or partial leg collapse towards midline or hind leg trembling during tail suspension. 2 = toes curl under at least twice during a 12-inch walk, or any part of foot is dragging along cage / table bottom. 3 = rigid paralysis or minimal joint movement, foot not being used for forward motion. 4 = humane endpoint, mouse cannot right itself within 30 seconds on either side. The locomotion of animals is quantified over a period of 46 hours (hrs) in the home cage, by automated sensing of body -heat image using an InfraMot (TSE-Systems).Individual animal movements are summed up every 30 minutes. Animal statistics: Homecage locomotion is analyzed using the 2-sided Student's t-test. Rotarod performance is evaluated using the 2-way ANOVA with repeated measures for each individual. Weight peak, weight decline, onset of symptoms and survival are calculated with GraphPad Prism 6 using the Logrank (Mantel-Cox) test. All other tests employ 2-way ANOVA.Survival

[0246] Animals in the survival study arm are followed until age day 150 or until the animals reach a humane end point criterion. The survival time of an animal is registered as the age day on which theWSGR Ref. No. : 64320-709.601 animal was euthanized due to disease model-related reasons i.e., clinical score 1.5 or more than 25% body weight loss. Spontaneous mortalities in the Test Laboratory in the current model are rare. Such cases are evaluated individually. In case significant weakness and body weight loss were observed prior to the mortality and in case no obvious non-disease related cause for the death is identified, the animal is included in the survival analysis. In case of non-disease-related mortalities, e.g., infection, tumor, injuries inflicted during handling or dosing, the animal is excluded from survival analysis but may be included in the assessment of functional outcomes.Euthanasia and Sample CollectionSurvival Study Arm

[0247] When reaching 150 days of age or earlier at the humane end point, the terminal body weight of the animals is recorded, and the animals are terminally anesthetized with pentobarbital (180 mg / kg, intraperitoneal (i.p. )) according to the Standard Operating Procedures (SOPs). The maximal volume of blood is collected in K3EDTA collection tubes, and animals decapitated. Terminal blood collection time is recorded. Blood samples are centrifuged at 2000 x g for 10 min at +4°C to separate a target of 200 pl of plasma. The volume and the color (clear, pink or red) of the separate plasma is recorded. The samples are frozen within a tube on top of dry ice and stored at -80°C until shipment for analysis.Sampling Study Arm

[0248] When reaching 115 days of age or earlier at the humane end point, the terminal body weight of the animals is recorded, and the animals terminally anesthetized with pentobarbital (180 mg / kg, i.p.) according to the Standard Operating Procedures (SOPs).Whole Blood Collection

[0249] After adequate anesthesia level has been confirmed, the maximal volume of blood is collected via cardiac puncture in 8 collection tubes, and animals decapitated.Hematology Sample

[0250] For hematology, 90 pl of the whole blood is aliquoted to separate, prelabeled K3EDTA collection tubes and maintained at +6 °C until used for hematology analysis within the next 24h.Plasma separation

[0251] The remaining whole blood (target: at least 400 pl) is split to a K3EDTA tube (target: -120 pl) and to a Li-hep tube (target: -280 pl). The blood samples are centrifuged at 2000 * g for 10 min at +4°C to separate plasma. The volumes and color (clear, pink, or red) of the separate plasma samples are recorded.Plasma samples for Luminex

[0252] For Luminex analysis, 35 pl of EDTA plasma is aliquoted in prelabeled (“plasma 1”) 2 ml Safelock Eppendorf tube (cat. no. 72.695.200 or equivalent), frozen on dry ice and stored at -80°C until analysis. For selected animals, another 35 pl aliquot might be collected to a separate vial, labeled as “plasma 2.”WSGR Ref. No. : 64320-709.601Plasma for clinical chemistry

[0253] For clinical chemistry, 60 pl of Lithium heparin (Li -hep) plasma is aliquoted to a separate 2 mL prelabeled (“plasma 3”) Safe-lock Eppendorf tube (CAT# 211-2165), volume recorded and frozen in liquid nitrogen and stored at -80°C until analysis.CSF for Neurofilament Light Chain (NfL) Analysis

[0254] CSF is collected (4-5 pl volume expected) and the quality of the samples evaluated and recorded (red / pink / clear). The samples are centrifuged at 18,800 x g for 10 min at +4°C and transferred to a sterile 1.5-ml Safe-lock Eppendorf tube with a micropipette, while avoiding touching the bottom of the tube or any pellet with the pipette tip. The volume of the final CSF is recorded. The CSF samples are snap-frozai on dry ice and stored at -80°C until NfL analysis.Perfusion

[0255] After blood and CSF collection, animals are transcardially perfused with ice-cold heparinized (Heparin 2.5 lU / mL) saline followed by tissue collection as stated below.Spinal Cord Cervical Segment for QUANTIGENE®

[0256] The spinal cord is dissected out and the cervical segment separated and weighed. A representative punch of 5-10 mg of tissue is collected, targeting the same site in every animal. (The absolute maximum weight of the collected sample is 25 mg.) Excess tissue is trimmed away primarily from the caudal end of the sample as needed. The final weight is recorded. The sample is placed in a prelabeled, round-bottom, nuclease-free 2-ml Safe-lock Eppendorf tube (cat. no. 72.695.200 or equivalent) filled with RNALATER® (Invitrogen, CAT# 10427114 or equivalent), in a minimum of 5X- 1 OX the volume of the tissue. The RNALATER® is removed after a 24-hour incubation at +4 °C, and the samples are stored at -80 °C until QUANTIGENE® sample processing and analysis.Spinal Cord Thoracic Segment for Luminex

[0257] The thoracic segment is collected, weighed and weight recorded (minimum of 20-30 mg of tissue for analysis). The sample is frozen in a prelabeled 2-ml Safe-lock Eppendorf tube (cat. no. 72.695.200 or equivalent) in liquid nitrogen and stored at -80 °C until tissue processing and analysis.Spinal Cord Lumbar Segment for IHC

[0258] Spinal cord Lumbar segment is separated, trimmed, and the caudal end of the sample marked with a small drop of tissue ink (FIG. 4). Only lumbar segments are collected for IHC and the sacral (S) segment is not collected. The sample is then placed in a tissue cassette on foam with a pre-cut groove to prevent the tissue from twisting during processing so that the caudal end faces the etiquette side of the cassette (FIG. 5). The sample is post-fixed in 4% paraformaldehyde (PF A) in 0. 1 M phosphate buffer (PB) in at least 50x tissue-to-fixative ratio for 24 hour at +4 °C with gentle rocking. For paraffin processing, the samples are washed once with lx PBS (pH 7.4) and stored for 0-5 days in lx PBS (pH 7.4) with 0.01% Na-Azide at +4 °C until paraffin-processed for IHC analysis.WSGR Ref. No. : 64320-709.601Vital organs collection and post-fixation

[0259] After transcardial perfusion, the whole brain, heart, lung, kidney, spleen and liver are collected, washed 3 times in 0. 1 M PB and post-fixed in at least lOx fixative- to-tissue volume of 10% neutral- buffered formalin (NBF) and stored at +4 °C. The samples are shipped in the fixative at +4 °C.Hematology and Clinical Chemistry

[0260] The following parameters are analyzed for the hematological analysis from whole blood: white blood cell count (WBC), red blood cell count (RBC), hematocrit (HCT), and mean corpuscular volume (MCV). The following parameters are analyzed from terminal plasma: creatinine, urea, total protein, AST, ALT, ALP, bilirubin, cholesterol, triglycerides and LDH. The clinical chemistry and hematological analysis are performed outside the Test Laboratory at Movet (Kuopio, Finland).QUANTIGENE® Gene Expression Assay

[0261] The expression of the specified 16 target genes (+5 suitable housekeeping genes for normalization; please see Table 13 for a 21plex) are examined in a representative piece of cervical spinal cord using a branched DNA (bDNA) assay (QUANTIGENE® Plex, Invitrogen).Tissue Processing for the QUANTIGENE® Assay

[0262] QuantiGene tissue lysate preparation (n = 30). The tissues (optimal: 5-10 mg pieces; max. 25 mg) are homogenized using the QUANTIGENE® Sample Processing Kit for Fresh or Frozen Tissues according to the manufacturer’s protocol using TissueLyser II (Qiagen). The prepared RNA-containing supernatants are stored at -80 °C until analysis.QUANTIGENE® Assay

[0263] The gene expression levels are determined using custom-prepared QUANTIGENE® Plex sets and a QUANTIGENE® Plex Assay Kit. The assay is performed according to the instructions provided by the manufacturer (Invitrogen). A small pilot is run separately before the actual assays to determine the optimal sample input to be used in the assays. Target expression levels are normalized using the geometrical mean of at least three different housekeeping genes where possible (Table 13). The housekeepers used are optimally stable in all the study conditions, as well as represent different expression levels (low, medium, medium / high).Table 13. Endogenous mouse genes to be measured by the QUANTIGENE® (bDNA) method.WSGR Ref. No. : 64320-709.601*TGF beta genes appeared more relevant in this context, so Tgfbl, Tgfb2 and Tgfb3 have now been included. **These housekeeping genes have been used in the SOD1 mouse previously; kept here, but need to control whether the test article affects these 3 genes / pathways. ***Actb and Gapdh added as per Sponsor's previous experience.Luminex Multiplex Protein Assay for Cytokines and Chemokines

[0264] The Luminex (multiplex protein) assay measuring the selected cytokines and chemokines is performed using the following samples: sampling / satellite study arm plasma (endpoint); n = 30 mice (aim: 30 pl of plasma / assessment); and sampling / satellite study arm cervical spinal cord (endpoint); n = 30 mice (aim: 20-30 mg of tissue).WSGR Ref. No. : 64320-709.601

[0265] The following 9 markers are analyzed using the MILLIPLEX® MAP Mouse Cytokine / Chemokine Magnetic Bead Panel - Immunology Multiplex Assay (Merck Millipore, cat. no. MCYTOMAG-70K): IL- 1 , IL-6, IL- 10, TNF-a, IFN-y, MCP-1 (monocyte chemoattractant protein 1 = chemokine (C-C motif) ligand 2 / CCL2), GM-CSF (granulocyte-macrophage colony stimulating factor), G-CSF (granulocyte colony stimulating factor, and VEGF-A (vascular endothelial growth factor).

[0266] The spinal cord tissue pieces are mechanically homogenized in a suitable lysis buffer to release soluble proteins, followed by collecting the protein-containing supernatants. Samples are aliquoted as needed to avoid repeated freeze-thaw cycles and stored at -80 °C until used in the LUMINEX® assay. Total protein concentrations will be measured, as needed.

[0267] Plasma samples are only be diluted for the assay as per the manufacturer’s recommendations, usually a dilution of 1 :2 (1 part plasma + 1 part diluent).

[0268] The assay is piloted before the analyses, and the exact protocol may change based on the piloting. The samples are analyzed as duplicates where possible. Beads are read on BIO-PLEX® 200 and results obtained by the BIO-PLEX® Manager software 6.2 (Bio-Rad Laboratories, Inc.). The software creates a standard curve for each analyte for inter- and extrapolating of the Net Mean Fluorescence Intensity (Net MFI) against the standard curve. The most suitable curve fitting for the data is used (using e.g, a 5-parameter logistic fit) for each analyte.Neurofilament Light Chain Analysis in CSF and Plasma Samples Using Quanterix Simoa Assay (NfL)

[0269] Neurofilament Light Chain (NfL) analysis from mouse CSF (n=30) and plasma (n=30) samples is performed in single determinations according to the QUANTERIX® SIMOA® NF-LIGHT™ Advantage PLUS Kit Assay (#104364) manufacturer’s protocol.Immunohistochemistry (IHQ: Tissue processing and paraffin embedding

[0270] The spinal cords are rinsed 2 times with 50% EtOH, 5 min for each rinse, before initiating paraffin processing. The fixed lumbar spinal cord segment is dehydrated and processed into paraffin blocks by applying ATP-50-2023 paraffin processing program in a tissue processing station (ASP300S, Leica Biosystems) with an ascending series of alcohol, followed by xylene and paraffin. The intended tissue processing program for the brains is shown in Table 14.Table 14. Tissue processing program for immunohistochemical analyses.WSGR Ref. No. : 64320-709.601RT = room temperature.Lumbar Spinal Cord Paraffin Embedding and Sectioning

[0271] L segments from three separate animals are embedded coronally in the same block. Caudal ends of the spinal cords will be marked using tissue ink. The spinal cords are embedded with the rostral end towards bottom of the mold (e.g., the face of the block / sectioning start site is the rostral part) (FIG. 6).Spinal Cord Sectioning

[0272] After paraffin embedding, the spinal cord segments (n=3 separate animals / block) are sectioned as 5-pm thick paraffin cross sections into six serial sections at a regular -700 pm intervals. A total of 9 series are generated per animal from the spinal cord. Series 1 is used for GFAP+Ibal double staining, Series 2 for BDNF staining, Series 3 for CHI3L1 staining, Series 4 for CX43 staining, Series 5 for misfolded SOD1 staining, Series 6 for Choline Acetyltransferase (ChAT) staining, series 7-8 are extra, back-up sections if staining needs to be repeated or additional staining for other markers performed) and series 9-10 are used for pilot staining and as negative controls. As the lumbar spinal cords are 5 mm - 8 mm when they are fresh, and spinal cords shrink during paraffin processing (25-30%), the 6th section is only collected if available.IHC Staining with Ventana Discovery Ultra

[0273] The immunostaining is performed with an automated IHC staining system (Ventana Discovery Ultra, Roche) on 5-pm-thick paraffin sections. Table 15 describes the used primary- and secondary antibodies and detection kits. In short, the protocol includes sequences of washes with reaction buffer (Cat # 950-300, Roche), antigen retrieval by CC1 solution (Cat # 950-500; 90 °C for 24 min), inhibitor of endogenous peroxidases (Discovery Inhibitor Cat # 760-4840 Roche), blocking of non-specific binding (10% NGS in PBST), addition of primary antibody and followed by addition of secondary antibody. The antigens are visualized using fluorescence / chromogenic detection. The denaturation step between any 2 sequential markers will be carried out by heating the samples at 100 °C in CC2 buffer (Cat #950-223) for about 30 min to prevent cross-reaction. After completion of the staining procedure, the glass slides will be removed from the automated staining system, washed and mounted with ROTI®Mount or PROLONG™ gold with DAPI and cover slips applied manually. The ideal dilutions / timings of primary / secondary antibodies and other experimental conditions are optimized during pilots. Based on pilot staining results chromogens / fluorochromes are chosen.WSGR Ref. No.: 64320-709.601Table 15. The details of primary antibodies and tissues.IHC Analysis

[0274] The stained sections are scanned with the Olympus VS 120 slide scanner and analyzed with VIS - Visiopharm Integrator System (Visiopharm, Denmark).Spinal Cord analysis:

[0275] Iba-1, GFAP, BDNF, CHI3L1, CX43 ChAT: ROI Lumbar spinal cord (whole spinal cord section) results are reported as positive staining percentage of region area (%):[%area = (stained area / total analyzed region area) * 100)].WSGR Ref. No. : 64320-709.601

[0276] Misfolded SOD1 Antibody (C4F6): ROI Lumbar spinal cord (ventral horn grey mater): results are reported as 1) Aggregates / mm2: All fluorescence above 3 pm2 is quantified as a true signal and signals below this threshold were considered as background and 2) Positive staining percentage of region area (%) [%area = (stained area / total analyzed region area) * 100)] .Statistical Analysis and Graphical Data Presentation

[0277] Prior to further statistical analysis, data quality check and validations are performed. During that process, potential outliers are identified and assessed. No outliers are removed from the data without a clear justification for removal (e.g, identified measurement error in the laboratory notes). Data are presented as pooled genders and males and females separately.

[0278] Statistical analysis is performed using the two-tailed Student's t-test with post-hoc Benjamin- Hochberg corrections and results are presented with standard deviations. qPCR data statistics for the human miRNA study is obtained using the DataAssist Software (LIFE TECHNOLOGIES®). Data comparisons are as follows: Groups 2 and 3 vs. Group 1 (therapy intervention effect).

[0279] Assumption of normality of each data set are primarily based on experience (e g, data within a population is known to be approximately Gaussian) and observations during validation phase. Some biological variables are known to follow lognormal distributions - in these cases, the data are first transformed to logarithms and then parametric statistical test can be used. The same normality assumption is used for series of experiments, or group of similar readouts from an assay. In addition, the D’Agostino-Pearson omnibus normality test can be used to support the decision on whether parametric or nonparametric test is used.

[0280] All values are presented as the mean ± standard error of the mean (SEM). In case of two or more comparisons per family, the multiplicity adjusted P values are presented. All statistical analyses are conducted with a significance level of a = 0.05, using GraphPad Prism (Version 9, GraphPad Software, Inc., San Diego, CA) or the R statistical software environment (R: A Language and Environment for Statistical Computing; R Core Team / R Foundation for Statistical Computing; Vienna, Austria; 2019; R- project.org). A summary of readouts for statistical analysis and visualization can be found in Table 16.Table 16. Summary of readouts for statistical analysis and visualizationWSGR Ref. No. : 64320-709.601

[0281] Results comprise body weights and clinical score over time, disease onset and survival, grip strength and Rotarod over time, Hematology and clinical chemistry at the end point, Luminex results, QuantiGene results, NfL results, IHC results, and Statistical analysis of the results.Table 17. Calculation of compound requirement

[0282] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of theWSGR Ref. No. : 64320-709.601 disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Ref. No.: 64320-709.601CLAIMSWhat is claimed:

1. A method of treating Amyotrophic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering human, neonatal cardiac mesenchymal progenitor cells (nMPCs), an nMPC secretome (conditioned medium), nMPC exosomes, an nMPC total secretome (total conditioned medium), or any combination thereof, whereby ALS is treated.

2. The method of claim 1, wherein the subject is administered the nMPCs.

3. The method of claim 2, wherein nMPCs are (a) positive for cell surface protein expression of c- kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4.

4. The method of any one of claims 1-3, wherein the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, or SOX2.

5. The method of any one of claims 1-4, wherein the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2.

6. The method of any one of claims 1-5, wherein the nMPCs are further negative for protein expression of pl6INK4aup to at least passage 8.

7. The method of any one of claims 1-6, wherein the nMPCs are (a) positive for protein expression ofc-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, or SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, or pl6INK4a.

8. The method of any one of claims 1-7, wherein the nMPCs are (a) positive for protein expression ofc-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a.

9. The method of any one of claims 1-8, wherein the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and / or HSF1.

10. The method of any one of claims 1-7, wherein the nMPCs are (a) positive for protein expression ofc-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a, and (c) genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1.

11. The method of any one of claims 1-10, wherein the nMPCs are genetically engineered to express or to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1.

12. The method of any one of claims 1-11, wherein the nMPCs are allogeneic.

13. The method of any one of claims 1-11, wherein the nMPCs are negative for:(i) a mutation in, or modified expression of, a SOD1 gene or RNA;WSGR Ref. No. : 64320-709.601(ii) a mutation in, or modified expression of, chromosome 9, open reading frame 72, optionally wherein the chromosome 9, open reading frame 72 comprises a C9ORF72 gene;(iii) a fused Fus gene mutation;(iv) a mutation in, or modified expression of, a TDP-43 gene or RNA;(v) a mutation in, or modified expression of, a STMN2 gene or RNA;(vi) a mutation in, or modified expression of, a UNC13A gene or RNA;(vii) a mutation in, or modified expression of, a TARDBP gene or RNA;(viii) a mutation in, or modified expression of, a hnRNPAl gene or RNA;(ix) a mutation in, or modified expression of, a hnRNPA2B 1 gene or RNA;(x) a mutation in, or modified expression of, &MATR3 gene or RNA;(xi) a mutation in, or modified expression of, an ANG gene or RNA;(xii) a mutation in, or modified expression of, a TUBA4A gene or RNA;(xiii) a mutation in, or modified expression of, an ANXA11 gene or RNA;(xiv) a mutation in, or modified expression of, a PRPH gene or RNA;(xv) a mutation in, or modified expression of, a DCTN1 gene or RNA;(xvi) a mutation in, or modified expression of, a PFN1 gene or RNA;(xvii) a mutation in, or modified expression of, a KIF5a gene or RNA;(xviii) a mutation in, or modified expression of, a UBQLN2 gene or RNA;(xix) a mutation in, or modified expression of, a SQSTM1 gene or RNA;(xx) a mutation in, or modified expression of, an OPTN gene or RNA;(xxi) a mutation in, or modified expression of, a VCP gene or RNA;(xxii) a mutation in, or modified expression of, a CHMP2B gene or RNA;(xxiii) a mutation in, or modified expression of, a VAPB / VAMP gene or RNA; or(xxiv) a mutation in, or modified expression of, a TBK1 gene or RNA;(xxv) a mutation in, or modified expression of, an EphA4 gene or RNA;(xxvi) a mutation in, or modified expression of, a NEK-1 gene or RNA; or(xxvii) a mutation in, or modified expression of, an ATXN2 gene or RNA.

14. The method of any one of claims 1-13, wherein the subject is administered the nMPC secretome.

15. The method of claim 14, wherein the nMPC secretome is positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, or Akt.

16. The method of claim 14 or 15, wherein the nMPC secretome is positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and Akt.

17. The method of any one of claims 1-16, wherein the nMPC secretome is positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of FUS, TAF15, CAP2, SNX9, PSMB7, SUM01, EIF3A, ECHI, HNRNPL, NTM, ANG, ERP44, NAA38, RRM2B, RAB1A, RAB1B, PGRMC1, ERH, TIGAR, ITGA2, SGTA, AR1C1, AKR1C2, DNASE2, HSP90AB4P, ANGPTL2, SF3B2, GREM1, ARF3, ARF1, ARF5, LSM5, PSMD9, SUGT1, ALDH1A3, PDGFRB,WSGR Ref. No.: 64320-709.601HIST1H1C, SP100, G6PD, PPIL1, SNRPD1, FDPS, RAB6B, RAB6A, DCTD, GLB1, ARIH1, IST1, HNRNPU, ULBP2, RAET1G, ERAP2, CDC42, EDF1, LIPG, FKBP4, RBMX, RBMXL1, RPL30, THY1, AP2M1, RPS16, FHL3, BZW1, BZW2, EIF4G1, PFDN4, CLTB, PIR, MVP, ECU, DCI, ALYREF, RPS10, PPP3CA, TAX1BP3, PPA1, DDX39B, DDX39A, GNPNAT1, LIMA1, SNRPD3, ADH5, ENAH, DYNLL1, DYNLL2, HUWE1, HMGB2, PTMS, UCHL3, PFDN2, DYNLRB1, HDGFRP2, INF2, HPRT1, EIF2S2, ADAMT7, EIF3C, EIF3CL, RPS5, VPS35, CS, ECHS1, UBQLN1, GBP2, GBP1, SF3B1, RANBP1, GNS, HLA-B, UBXN1, GNPDA1, IGF2, EIF3B, TCEB1, VASP, GSR, HLA-A, HLA-HIGF2, NUDT5, COPE, HNRNPA3, SRI, HNRNPC, MMP10, EWSR1, IPO7, YARS, TMPO, HNRNPH1, HLA-A, PDGFC, IL1B, S100A10, TXNDC2, DPY30, LAMP1, LYPLA1, CBR1, ARHGAP1, TPR, NAMPT, NAMPTL, CXCL2, EIF4A2, RPS4X, PABPN1, PHLDB1,NAP1L4, DDX6, PSMC3, MAP7D1, OSCAR, TES, NMT1,NMT2, MAT2A, PRMT1, COX17, SELM, CARS, PPP2CA, PPP2CB, VEGFC, VPS26A, PXN, PAWR, STRAP, RAB11A, RAB11B, EIF5, COPZ1, COPS3, CHMP4B, PDXK, RALA, RALB, SEC13, FHL2,TOM1, PYCARD, PDLIM4, RBBP4, RBBP7, RPS20, SRSF1, DLD, SNRPE, EIF4EBP1, RPLPO, RPLP0P6, LSM3, SLC16A3, CSE1L, NUCKS1, TSNAX, RPSA, RPSAP58, AP1B1, EIF2S1, LCP1, PSMD4, AARS, SRSF2, SFRS2, TRIP10, CLTA, USO1, VBP1, UBE2K, MRPL12, UAP1, HIST1H1B, YBX3, RPL10A, IDH1, SNRPN, SNRPB, PCK2, EIF6, MMP3, DHX9, MATR3, UBE2D2, UBE2D3, PTRF, ACTR1A, LAMA1, PDCD6IP, QARS, HN1L, PLBD2, OS9, ILF3, ISOCI, PPP1R7, MAPK1, PDCD1LG2, GOLGB1, HSPA9, ACBD3, BTF3, UBE2I, SRRT, EIF4H, FERMT2, EIF3G, PARVA, DBNL, BOLA2, MAPRE1, PRDX5, STX12, EIF1, SMS, GRPEL1, OAF, SYNCRIP, ECE1, VAPA, PCNP, PSME1, EIF3J, C14orfl66, DDX1, AKR1B1, SRP9, PCMT1, XPO1, MMP9, RPS7, NRCAM, FKBP3, SSB, DSC3, XRCC5, CCT6A, LIF, SERPINA9, SND1, ANP32B, G3BP1, GPC6, GLOD4, RRBP1, TGFB1, USP14, PLAUR, CXCL3, FASN, COPB2, TARDBP, TNFAIP6, RPS3, NDUFAB1, LMNB1, COL16A1, PSMF1, PHGDH, PRKCDBP, CXCL5, YAP1, TROVE2, MACF1, EIF5B, STX7, PFDNS, APOA1, PDAP1, STX12, EIF3K, FH, RBM8A, EIF1, RPS2, PTGR1, EEA1, CAB39, DCTN2, GDF15, MYL9, TCP1, RPS12, RAD23A, SKP1, PDLIM1, HSPH1, PSMB3, SUB1, API5, TWF2, KTN1, DYNC1H1, SNRNP70, MCFD2, PPP1R18, PTGES3, APEX1, TNKS1BP1, FUBP1, VAT1, PAFAH1B1, CBX1, PTK7, HNRPDL, ZNF185, DYNC1I2, KHDRBS1, SFPQ, PDCD5, TRIM28, PPIC, NARS, PCBP2, CD2AP, PSMA1, CAPRIN1, KHSRP, NSFL1C, PPP1R12A, NUDC, MFAP2, IGF2R, RGMB, PABPC1, MAN1A1, DDB1, PTBP1, FBN2, CNBP, OTUB1, NASP, HSPD1, EEF1D, RNPEP, CRKL, HNRNPAB, EIF3F, PAICS, ILF2, COPB1, PA2G4, COPA, NT5E, HMGA1, SF1, XRCC6, GNB2L1, MEI, MTAP, CCT2, ARCN1, PEPD, and EPB41L3.

18. The method of any one of claims 1-17, wherein the nMPC secretome is positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of PPP1CA, PPP1CC, STI 3. ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8-MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP,WSGR Ref. No.: 64320-709.601NAP1L1, HSP90AA1, SET, ENO1, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, 42254, and PAFAH1B2.

19. The method of any one of claims 1-17, wherein the nMPC secretome is positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of PPP1CA, PPP1CC, STI 3. ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8-MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP, NAP1L1, HSP90AA1, SET, ENO1, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, and PAFAH1B2.

20. The method of any one of claims 1-19, wherein a symptom of ALS is improved by 2% or more, or by 2-fold or more, following treatment as compared to treatment with a placebo or compared to the subject prior to treatment.

21. The method of claim 20, wherein the symptom comprises an elevated level of Interleukin (IL) - 1 P, IL-6, IL-8, IL-12, IL-17a, IL-18, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-a, interferon gamma (IFN-y), or a combination thereof.

22. The method of any one of claims 1-21, wherein administration of the allogeneic, human, neonatal cardiac mesenchymal progenitor cells (nMPCs), an nMPC secretome (conditioned medium), nMPC exosomes, an nMPC total secretome (total conditioned medium), or any combination thereof, inhibits IL- ip, IL-6, IL-8, IL-12, IL-17a, IL-18, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-a, interferon gamma (IFN-y), or a combination thereof by at least 2% or by at least 2-fold as compared to treatment with a placebo or compared to the subject prior to treatment.

23. The method of any one of claims 1-22, wherein the subject exhibits an improvement in shortness of breath (e.g, difficulty breathing), fatigue, muscle weakness (e.g, feeling tired, even after resting), muscle twitching (e.g., twitching, cramping, or spasms in the muscles of the hands, feet, arms, shoulders, tongue, etc.), speech difficulty (e.g., slurred, nasal, or thick speech, difficulty projecting the voice, etc.), difficulty swallowing, difficulty chewing, trouble walking (e.g, tripping, falling, etc.), uncontrollable periods of laughing, uncontrollable periods of crying, uncontrollable periods of loss of motor control (e.g., drooling, difficulty raising the foot, clumsiness, dropping things, paralysis, constipation, etc.), or a or a combination thereof, following administration of allogeneic, human, neonatal cardiac mesenchymal progenitor cells (nMPCs), a secretome (conditioned medium) produced by the nMPCs, exosomes produced by the nMPCs, or any combination thereof, compared to administration of a placebo or compared to the subject prior to treatment.

24. The method of any one of claims 20-23, wherein the symptom further comprises neurodegeneration, inflammation, oxidative stress, or a combination thereof.WSGR Ref. No.: 64320-709.60125. The method of any one of the preceding claims, wherein treatment partially or completely reduces neurodegeneration.

26. The method of any one of claims 1-25, wherein treatment reduces neurodegeneration by about 2% or by at least 2-fold compared to treatment with a placebo or compared to the subject prior to administration of the nMPCs.

27. The method of any one of the preceding claims, wherein treatment reduces inflammation by at least 2% or by at least 2-fold as compared to treatment with a placebo or compared to the subj ect prior to treatment.

28. The method of any one of the preceding claims, wherein treatment reduces oxidative stress by at least 2% or by at least 2-fold as compared to treatment with a placebo or compared to the subj ect prior to treatment.

29. The method of any one of the preceding claims, wherein administration comprises an intravenous (IV) injection, an intravenous (IV) catheter (e.g, intravenous (IV) infusion), a subcutaneous injection, or a parenteral injection.

30. The method of any one of the preceding claims, wherein the nMPCs are administered by intravenous infusion.

31. The method of any one of the preceding claims, wherein the nMPCs are administered to the subject once every twenty-eight (28) days.

32. The method of any one of the preceding claims, wherein the nMPCs are administered to the subject for from about 1 month to about 100 years.

33. The method of any one of the preceding claims, wherein the nMPCs are non-naturally occurring.

34. The method of any one of the preceding claims, wherein the nMPCs are not xenogenic.

35. The method of any one of the preceding claims, wherein the nMPCs are infused via IV catheter for about 60 minutes to about 180 minutes.

36. The method of any one of the preceding claims, wherein the subject is administered a first dose of the nMPCs at day 0 and a second dose of the nMPCs at day 28.

37. The method of any one of the preceding claims, wherein the method further comprises administering to the subject an additional therapeutic agent or therapy.

38. The method of claim 37, wherein the one or more additional therapeutic agent(s) comprise(s) Riluzole, Edaravone, Tofersen, Nuedexta, sodium phenylbutyrate-taurursodiol (PB-TUDCA), or a combination thereof.

39. The method of any one of the preceding claims, wherein the subject is a mammal.

40. The method of claim 39, wherein the mammal is a human.

41. The method of claim 40, wherein the human is a child of from birth to 18 years of age.

42. The method of claim 40, wherein the human is an adult of 18 years of age or older.

43. The method of any one of the previous claims, further comprising administering nMPC exosomes.WSGR Ref. No.: 64320-709.60144. The method of claim 43, wherein the nMPC exosomes are positive for protein expression of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and / or Akt.

45. The method of claim 42 or 43, wherein the nMPC exosomes are positive for expression of CD81 and / or CD63.

46. The method of any one of claims 43-45, wherein the nMPC exosomes are negative for protein expression of IGF1.

47. The method of any one of claims 43-46, wherein the nMPC exosomes comprise particles in a range of from about 50 nm to about 1000 nm.

48. The method of any one of claims 43-47, wherein the nMPC exosomes comprise particles in a range of from about 100 nm to about 120 nm.

49. The method of any one of the preceding claims, wherein the subj ect is screened for a symptom at one or more times prior to treatment, during treatment, and / or after treatment.

50. The method of claim 49, wherein screening comprises a physical evaluation, a medical history review, or a diagnostic test.

51. The method of claim 49 or 50, wherein the diagnostic test comprises an electromyography (EMG), a nerve conduction study, a blood test, a urine test, or an MRI scan.

52. The method of any one of the preceding claims, wherein the subject is assessed at day 7 following treatment.

53. The method of any one of the preceding claims, wherein one or more proinflammatory cytokines in the subject are reduced by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

54. The method of claim 53, wherein the one or more proinflammatory cytokines comprise IL-1 , IL-6, IL-8, IL-12, IL-17a, IL-18, MCP-1, TNF-a, IFN-y, or a combination thereof.

55. The method of claim 54, wherein the one or more proinflammatory cytokines comprise IL-10, IL-6, IL-8, IL-12, IL-17a, IL-18, MCP-1, TNF-a, and IFN-y.

56. The method of claim 54, wherein the one or more proinflammatory cytokines comprise IL-8, IL- 173, MCP-1, TNF-a, and IFN-y.

57. The method of claim 54, wherein the one or more proinflammatory cytokines comprise IL-8, MCP-1, and TNF-a.

58. The method of claim 54, wherein the one or more proinflammatory cytokines comprise IL-17a and IFN-y.

59. The method of any one of the preceding claims, wherein the subject has an improved quality of life or an improved length of life following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

60. The method of any one of the preceding claims, wherein a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is above about 10 pg / ml, about 20 pg ml, about 30 pg / ml, about 40 pg / ml, or about 50 pg / ml prior to treatment.WSGR Ref. No.: 64320-709.60161. The method of any one of the preceding claims, wherein a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is reduced by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

62. The method of any one of the preceding claims, wherein a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream of the subject is reduced by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

63. The method of any one of the preceding claims, wherein a level of Neurofilament Light Chain (NfL) protein fragment in the cerebrospinal fluid (CSF) of the subject is reduced by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

64. The method of any one of the preceding claims, wherein a level of Neurofilament Light Chain (NfL) protein fragment in the bloodstream or cerebrospinal fluid (CSF) of the subject is reduced by at least about 1 pg / ml, 2 pg / ml, 3 pg / ml, 4 pg / ml, 5 pg / ml, 6 pg / ml, 7 pg / ml, 8 pg / ml, 9 pg / ml, 10 pg / ml, about 20 pg ml, about 30 pg / ml, about 40 pg / ml, or about 50 pg / ml following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

65. The method of any one of the preceding claims, wherein a level of interleukin 8 (IL -8), lipopolysaccharide binding protein (LBP), cluster of differentiation 14 (CD14), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), Insulin-like Growth Factor 1 (IGF-1), Epidermal Growth Factor (EGF), Transforming Growth Factor Beta 1 (TGF -pi), TGF- 02, TGF-03, neurotrophin-3 (NTF-3), hepatocyte growth factor (HGF), growth differentiation factor 15 (GDF-15), heparin-binding epidermal growth factor-like growth factor (HB-EGF), chitinase-3-like protein 1 (CHI3L1) (Gp39), connexin 39 (Cx39), Cyclophilin B (CypB) (PPIB), TATA-Binding Protein (TBP), DNA-directed RNA polymerase II subunit RPB1 (POLR2A), Glyceraldehyde 3 -Phosphate Dehydrogenase (GAPDH), beta actin (ACTB), or a combination thereof of the subject is modulated by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

66. The method of any one of the preceding claims, wherein a level of IL -8, LBP, CD 14, BDNF, GDNF, IGF-1, TGF-01, TGF-02, TGF-03, GDF-15, HB-EGF, CHI3L1 (Gp39), Cx39, PPIB, ACTB, or a combination thereof of the subject is reduced by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

67. The method of any one of the preceding claims, wherein a level of EGF, PPIB, GAPDH, or a combination thereof of the subject is increased by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

68. The method of any one of the preceding claims, wherein a level of interleukin 10 (IL-10), IL-6, IL- 10, TNF-a, or a combination thereof of the subject is modulated by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

69. The method of any one of the preceding claims, wherein a level of Phosphorylated Neurofilament Heavy Chain (pNfH), TDP-43, FUS, SOD1, a dipeptide repeat (DPR) protein, tau protein,WSGR Ref. No. : 64320-709.601 phosphorylated tau protein (p-tau), clusterin, a chitinase-like protein, creatine kinase (CK), Glial Fibrillary Acidic Protein (GFAP), or a combination thereof of the subject is modulated by at least 2% compared to treatment with a placebo or compared to the subject prior to treatment.

70. The method of any one of the preceding claims, wherein a level of Phosphorylated Neurofilament Heavy Chain (pNfH), TDP-43, FUS, SOD1, a dipeptide repeat (DPR) protein, tau protein, phosphorylated tau protein (p-tau), clusterin, a chitinase-like protein, creatine kinase (CK), Glial Fibrillary Acidic Protein (GFAP), or a combination thereof of the subject is reduced by at least 2% following treatment compared to treatment with a placebo or compared to the subject prior to treatment.

71. The method of any one of the preceding claims, wherein the subject is biologically a male.

72. The method of any one of the preceding claims, wherein the subject is biologically a female.

73. The method of any one of the preceding claims, wherein the subject is from about 1 to about 10 years of age, from about 11 to about 20 years of age, from about 21 to about 30 years of age, from about 31 to about 40 years of age, from about 51 to about 60 years of age, from about 71 to about 80 years of age, from about 81 to about 90 years of age, or about 91+ years of age.

74. The method of any one of the preceding claims, wherein the nMPCs are stable in storage at a temperature for a period of time.

75. The method of claim 74, wherein the temperature is about 4 °C, 1 °C, 0 °C, -10 °C, -20 °C, or - 80 °C.

76. The method of claim 74 or 75, wherein the period of time is at least about 1, 2, 3, 4, 5, 6, or 7 days; at least about 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

77. The method of any one of claims 74-76, wherein the nMPCs are stored in cell cry opreservation media-CSIO (CRYOSTOR®; Sigma- Aldrich, Israel).

78. The method of any one of claims 74-77, wherein the nMPCs are thawed in a 37 °C water bath until a single ice crystal remained prior to administration to the subject.

79. The method of claim 78, wherein the nMPCs are further washed with a volume of Complete Medium (CM) prior to administration to the subject.

80. The method of claim 79, wherein the nMPCs are washed twice with a volume of Complete Medium (CM) prior to administration to the subject.

81. The method of claim 78 or 79, wherein the volume to be administered is at least about 10 ml.

82. The method of any one of the preceding claims, wherein the nMPCs are:(a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, or 8 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5;(b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4;(c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / orWSGR Ref. No.: 64320-709.601(d) negative for protein expression of pl6INK4aup to at least passage 8.

83. The method of any one of the preceding claims, wherein the nMPCs are:(a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and(b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a.

84. The method of any one of claims 1-83, wherein the ALS is familial ALS.

85. The method of any one of claims 1-84, wherein the secretome does not comprise 42254.

86. The method of any one of claims 1-85, wherein the subject is administered an intravenous (IV) bag comprising a total of from about 50 million to about 250 million nMPCs.

87. The method of claim 86, wherein the subj ect is administered one or more IV bags comprising the nMPCs.

88. The method of claim 86 or 87, wherein the subject is administered more than one IV bag, and each bag is administered at about every 28 days.

89. A composition comprising allogeneic, human nMPCs, wherein the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4.

90. The composition of claim 89, wherein the nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2.

91. The composition of claim 89 or 90, wherein the nMPCs are further negative for protein expression of p!6INK4aup to at least passage 8.

92. The composition of any one of claims 89-91, wherein the nMPCs are genetically engineered.

93. The composition of claim 92, wherein the nMPCs are genetically engineered to overexpress one or more paracrine factors.

94. The composition of claim 92 or 93, wherein the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, or HSF1.

95. The composition of claim 94, wherein the nMPCs are genetically engineered to overexpress 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 of HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1.

96. The composition of claim 94 or 95, wherein the nMPCs are genetically engineered to overexpress HGF, SCF, SDF-la, ANG1, VEGFA, PDGFB, bFGF, HSP20, Akt, and HSF1.

97. The composition of any one of claims 89-96, wherein the nMPCs are non-naturally occurring.

98. The composition of any one of claims 89-97, wherein the nMPCs are not xenogenic.

99. The composition of any one of claims 89-98, wherein the nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, andNkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4.

100. The composition of any one of claims 89-99, wherein the nMPCs are negative for:WSGR Ref. No. : 64320-709.601(i) a mutation in, or modified expression of, a SOD1 gene or RNA;(ii) a mutation in, or modified expression of, chromosome 9, open reading frame 72, optionally wherein the chromosome 9, open reading frame 72 comprises a C9ORF72 gene;(iii) a fused Fus gene mutation;(iv) a mutation in, or modified expression of, a TDP-43 gene or RNA;(v) a mutation in, or modified expression of, a STMN2 gene or RNA;(vi) a mutation in, or modified expression of, a UNC13A gene or RNA;(vii) a mutation in, or modified expression of, a TARDBP gene or RNA;(viii) a mutation in, or modified expression of, a hnRNPAl gene or RNA;(ix) a mutation in, or modified expression of, a hnRNPA2B 1 gene or RNA;(x) a mutation in, or modified expression of, &MATR3 gene or RNA;(xi) a mutation in, or modified expression of, an ANG gene or RNA;(xii) a mutation in, or modified expression of, a TUBA4A gene or RNA;(xiii) a mutation in, or modified expression of, an ANXA11 gene or RNA;(xiv) a mutation in, or modified expression of, a PRPH gene or RNA;(xv) a mutation in, or modified expression of, a DCTN1 gene or RNA;(xvi) a mutation in, or modified expression of, a PFN1 gene or RNA;(xvii) a mutation in, or modified expression of, a KIF5a gene or RNA;(xviii) a mutation in, or modified expression of, a UBQLN2 gene or RNA;(xix) a mutation in, or modified expression of, a SQSTM1 gene or RNA;(xx) a mutation in, or modified expression of, an OPTN gene or RNA;(xxi) a mutation in, or modified expression of, a VCP gene or RNA;(xxii) a mutation in, or modified expression of, a CHMP2B gene or RNA;(xxiii) a mutation in, or modified expression of, a VAPB / VAMP gene or RNA; or(xxiv) a mutation in, or modified expression of, a TBK1 gene or RNA;(xxv) a mutation in, or modified expression of, an EphA4 gene or RNA;(xxvi) a mutation in, or modified expression of, a NEK-1 gene or RNA; or(xxvii) a mutation in, or modified expression of, an ATXN2 gene or RNA.

101. The composition of any one of claims 89-100, wherein the conditioned medium or exosomes are positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of FUS, TAF15, CAP2, SNX9, PSMB7, SUMO1, EIF3A, ECHI, HNRNPL, NTM, ANG, ERP44, NAA38, RRM2B, RAB1A, RAB1B, PGRMC1, ERH, TIGAR, ITGA2, SGTA, AR1C1, AKR1C2, DNASE2, HSP90AB4P, ANGPTL2, SF3B2, GREM1, ARF3, ARF1, ARF5, LSM5, PSMD9, SUGT1, ALDH1A3, PDGFRB, HIST1H1C, SP100, G6PD, PPIL1, SNRPD1, FDPS, RAB6B, RAB6A, DCTD, GLB1, ARIH1, IST1, HNRNPU, ULBP2, RAET1G, ERAP2, CDC42, EDF1, LIPG, FKBP4, RBMX, RBMXL1, RPL30, THY1, AP2M1, RPS16, FHL3, BZW1, BZW2, EIF4G1, PFDN4, CLTB, PIR, MVP, ECU, DCI, ALYREF, RPS10, PPP3CA, TAX1BP3, PPA1, DDX39B, DDX39A, GNPNAT1, LIMA1,WSGR Ref. No.: 64320-709.601SNRPD3, ADH5, ENAH, DYNLL1, DYNLL2, HUWE1, HMGB2, PTMS, UCHL3, PFDN2, DYNLRB1, HDGFRP2, INF2, HPRT1, EIF2S2, ADAMT7, EIF3C, EIF3CL, RPS5, VPS35, CS, ECHS1, UBQLN1, GBP2, GBP1, SF3B1, RANBP1, GNS, HLA-B, UBXN1, GNPDA1, IGF2, EIF3B, TCEB1, VASP, GSR, HLA-A, HLA-HIGF2, NUDT5, COPE, HNRNPA3, SRI, HNRNPC, MMP10, EWSR1, IPO7, YARS, TMPO, HNRNPH1, HLA-A, PDGFC, IL1B, S100A10, TXNDC2, DPY30, LAMP1, LYPLA1, CBR1, ARHGAP1, TPR, NAMPT, NAMPTL, CXCL2, EIF4A2, RPS4X, PABPN1, PHLDB1, NAP1L4, DDX6, PSMC3, MAP7D1, OSCAR, TES, NMT1, NMT2, MAT2A, PRMT1, COX17, SELM, CARS, PPP2CA, PPP2CB, VEGFC, VPS26A, PXN, PAWR, STRAP, RAB11A, RAB11B, EIF5, COPZ1, COPS3, CHMP4B, PDXK, RALA, RALB, SEC13, FHL2, TOMI, PYCARD, PDLIM4, RBBP4, RBBP7, RPS20, SRSF1, DLD, SNRPE, EIF4EBP1, RPLPO, RPLP0P6, LSM3, SLC16A3, CSE1L, NUCKS1, TSNAX, RPSA, RPSAP58, AP1B1, EIF2S1, LCP1, PSMD4, AARS, SRSF2, SFRS2, TRIP10, CLTA, USO1, VBP1, UBE2K, MRPL12, UAP1, HIST1H1B,YBX3, RPL10A, IDH1, SNRPN, SNRPB, PCK2, EIF6, MMP3, DHX9, MATR3, UBE2D2, UBE2D3, PTRF, ACTR1A, LAMA1, PDCD6IP, QARS, HN1L, PLBD2, OS9, ILF3, ISOCI, PPP1R7, MAPK1, PDCD1LG2, GOLGB1, HSPA9, ACBD3, BTF3, UBE2I, SRRT, EIF4H, FERMT2, EIF3G, PARVA, DBNL, BOLA2, MAPRE1, PRDX5, STX12, EIF1, SMS, GRPEL1, OAF, SYNCRIP, ECE1, VAPA, PCNP, PSME1, EIF3J, C14orfl66, DDX1, AKR1B1, SRP9, PCMT1, XPO1, MMP9, RPS7, NRCAM, FKBP3, SSB, DSC3, XRCC5, CCT6A, LIF, SERPINA9, SND1, ANP32B, G3BP1, GPC6, GLOD4, RRBP1, TGFB1, USP14, PLAUR, CXCL3, FASN, COPB2, TARDBP, TNFAIP6, RPS3, NDUFAB1, LMNB1, COL16A1, PSMF1, PHGDH, PRKCDBP, CXCL5, YAP1, TROVE2, MACF1, EIF5B, STX7, PFDNS, APOA1, PDAP1, STX12, EIF3K, FH, RBM8A, EIF1, RPS2, PTGR1, EEA1, CAB39, DCTN2, GDF15, MYL9, TCP1, RPS12, RAD23A, SKP1, PDLIM1, HSPH1, PSMB3, SUB1, API5, TWF2, KTN1, DYNC1H1, SNRNP70, MCFD2, PPP1R18, PTGES3, APEX1, TNKS1BP1, FUBP1, VAT1, PAFAH1B1, CBX1, PTK7, HNRPDL, ZNF185, DYNC1I2, KHDRBS1, SFPQ, PDCD5, TRIM28, PPIC, NARS, PCBP2, CD2AP, PSMA1, CAPRIN1, KHSRP, NSFL1C, PPP1R12A, NUDC, MFAP2, IGF2R, RGMB, PABPC1, MAN1A1, DDB1, PTBP1, FBN2, CNBP, OTUB1, NASP, HSPD1, EEF1D, RNPEP, CRKL, HNRNPAB, EIF3F, PAICS, ILF2, COPB1, PA2G4, COPA, NT5E, HMGA1, SF1, XRCC6, GNB2L1, MEI, MTAP, CCT2, ARCN1, PEPD, and EPB41L3.

102. The composition of any one of claims 89-101, wherein the nMPC secretome is positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of PPP1CA, PPP1CC, ST 13. ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8- MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP, NAP1L1, HSP90AA1, SET, ENO1, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, 42254, and PAFAH1B2.WSGR Ref. No.: 64320-709.601103. The composition of any one of claims 89-102, wherein the nMPC secretome is positive for protein expression of one, two, three, four, five, six, seven, eight, nine, ten, or more of PPP1CA, PPP1CC, ST13, ST13P5, ST13P4, HMGB1, HMGB1P1, HNRNPA1, HNRNPA1L2, NEDD8, N EDD8- MDP1, YBX1, YBX2, CCT7, HNRNPH3, LTBP1, UBE2N, IL8, CYCS, LGALS1, IL6, PGAM1, YWHAH, TXN, ARHGDIB, PSMA3, RAN, TPM3, BASP1, HSPA8, ITGAV, PPIA, NACA, HSP90AB1, PCBP1, CAP1, NUTF2, PSMA5, CAPZA1, RPLP1, GLO1, ACTG1, ICAM1, SDC4, SOD2, PSMB4, TFPI2, C1QBP, NAP1L1, HSP90AA1, SET, ENO1, CCT3, GOT2, HSPE1, PGM3, PLIN3, CNDP2, PGD, SH3BGRL, TLN1, HSP90AB2P, GOT1, HNRNPAO, FTH1, TPD52L2, UGP2, AHNAK, CCT8, PSMA7, CCT4, SRPX2, CLIC4, KPNB1, AP2B1, and PAFAH1B2.

104. The composition of any one of claims 89-103, wherein the cells are obtained from a subject who is biologically a female.

105. The composition of any one of claims 89-104, wherein the cells are obtained from a subject who is biologically a male.

106. The composition of any one of claims 89-105, wherein the nMPCs are:(a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, or 8 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5;(b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4;(c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / or(d) negative for protein expression of pl6INK4aup to at least passage 8.

107. The composition of any one of claims 89-106, wherein the nMPCs are:(a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and(b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a.

108. The composition of any one of claims 89-107, wherein the composition further comprises a nMPC secretome, a nMPC exosome, or a nMPC total conditioned secretome.

109. The composition of claim 108, wherein the composition further comprises the nMPC total secretome (total conditioned medium).

110. The composition of claim 108 or 109, wherein the nMPC secretome, the nMPC exosome, or the nMPC total secretome (total conditioned medium) are positive for protein expression of HGF, SCF, SDF- la, ANG1, VEGFA, PDGFB, bFGF, HSP20, and / or Akt.

111. The composition of any one of claims 108-110, wherein the nMPC secretome, the nMPC exosome, or the nMPC total secretome (total conditioned medium) are negative for protein expression of IGF1.WSGR Ref. No. : 64320-709.601112. The composition of any one of claims 108-111, wherein the nMPC exosomes comprise particles in a range of from about 50 nm to about 1000 nm.

113. The composition of any one of claims 108-112, wherein the nMPC exosomes comprise particles in a range of from about 100 nm to about 120 nm.

114. A composition comprising cryopreserved, allogeneic, human nMPCs, wherein the cryopreserved, allogeneic, human nMPCs are (a) positive for protein expression of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and / or Nkx2.5 and (b) negative for protein expression of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and / or SSEA4.

115. The composition of claim 113, wherein the cryopreserved, allogeneic, human nMPCs are further positive for protein expression of PDL1, OCT3 / 4, NANOG, KLF4, and / or SOX2.

116. The composition of claim 113 or 115, wherein the cryopreserved, allogeneic, human nMPCs are further negative for protein expression of pl6INK4aup to at least passage 8.

117. The composition of any one of claims 113-116, wherein the composition further comprises a cryopreserved nMPC secretome or cryopreserved exosomes.

118. The composition of claim 117, wherein the composition further comprises the cryopreserved nMPC secretome and cryopreserved exosomes.

119. The composition of any one of claims 113-118, wherein the nMPCs are:(a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, or 8 of c-kit (CD117), Ki67, CD44, CD73, CD47, CD105, CD90, and Nkx2.5;(b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, and SSEA4;(c) positive for protein expression of 1, 2, 3, 4, or 5 of PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and / or(d) negative for protein expression of pl6INK4aup to at least passage 8.

120. The composition of any one of claims 113-119, wherein the nMPCs are:(a) positive for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of c-kit (CD117), CD44, CD73, CD47, Ki67, CD105, CD90, Nkx2.5, PDL1, OCT3 / 4, NANOG, KLF4, and SOX2; and(b) negative for protein expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CD34, CD45, CD31, PECAM-1, tryptase, GATA4, ISL1, SSEA3, SSEA4, and pl6INK4a.

121. The composition of any one of claims 113-120, wherein the nMPCs do not express 42254.

122. The composition of any one of claims 101-112, wherein the nMPCs are negative for:(i) a mutation in, or modified expression of, a SOD1 gene or RNA;(ii) a mutation in, or modified expression of, chromosome 9, open reading frame 72, optionally wherein the chromosome 9, open reading frame 72 comprises a C9ORF72 gene;(iii) a fused Fus gene mutation;(iv) a mutation in, or modified expression of, a TDP-43 gene or RNA;(v) a mutation in, or modified expression of, a STMN2 gene or RNA;WSGR Ref. No. : 64320-709.601(vi) a mutation in, or modified expression of, a UNC13A gene or RNA;(vii) a mutation in, or modified expression of, a TARDBP gene or RNA;(viii) a mutation in, or modified expression of, a hnRNPAl gene or RNA;(ix) a mutation in, or modified expression of, a hnRNPA2B 1 gene or RNA;(x) a mutation in, or modified expression of, & MATR3 gene or RNA;(xi) a mutation in, or modified expression of, an ANG gene or RNA;(xii) a mutation in, or modified expression of, a TUBA4A gene or RNA;(xiii) a mutation in, or modified expression of, an ANXA11 gene or RNA;(xiv) a mutation in, or modified expression of, a PRPH gene or RNA;(xv) a mutation in, or modified expression of, a DCTN1 gene or RNA;(xvi) a mutation in, or modified expression of, a PFN1 gene or RNA;(xvii) a mutation in, or modified expression of, a KIF5a gene or RNA;(xviii) a mutation in, or modified expression of, a UBQLN2 gene or RNA;(xix) a mutation in, or modified expression of, a SQSTM1 gene or RNA;(xx) a mutation in, or modified expression of, an OPTN gene or RNA;(xxi) a mutation in, or modified expression of, a VCP gene or RNA;(xxii) a mutation in, or modified expression of, a CHMP2B gene or RNA;(xxiii) a mutation in, or modified expression of, a VAPB / VAMP gene or RNA; or(xxiv) a mutation in, or modified expression of, a TBK1 gene or RNA;(xxv) a mutation in, or modified expression of, an EphA4 gene or RNA;(xxvi) a mutation in, or modified expression of, a NEK-1 gene or RNA;(xxvii) a mutation in, or modified expression of, an ATXN2 gene or RNA.

123. The composition of any one of claims 113-122, wherein the nMPCs are cryopreserved in a cryopreservation medium.

124. The composition of claim 123, wherein the cry opreservation medium comprises a permeating cryoprotective agent.

125. The composition of claim 124, wherein the permeating cryoprotective agent comprises a US Pharmacopeia (USP)-grade permeating cryoprotective agent.

126. The composition of claim 124 or 125, wherein the cryopreservation medium comprises about 10% of the permeating cryoprotective agent.

127. The composition of any one of claims 123-126, wherein the cryopreservation medium comprises about 10% of a Dimethyl Sulfoxide (DMSO).

128. The composition of any one of claims 123-127, wherein the cryopreservation medium comprises a US Pharmacopeia (USP)-grade DMSO.

129. The composition of any one of claims 123-128, wherein the cryopreservation medium is serum- free, protein-free, sterile, free of toxins, GMP certified, free of endotoxins, or free of pathogens.

130. A unit dose of the composition of any one of claims 89-129.

131. An intravenous (IV) bag comprising the composition of any one of claims 89-129.WSGR Ref. No.: 64320-709.601132. The IV bag of claim 131, wherein the IV bag comprises a total of from about 50 million to about250 million cells.

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