Methods for the generation and preparation of human immature dental pulp stem cell-derived exosomes (NESTAEXO) for therapeutic applications

Abstract

The present invention relates to a scalable method for isolating exosomes from human immature dental pulp stem cell (hIDPSC) cultures. The present invention also provides pharmaceutical compositions comprising the exosomes and methods of using these pharmaceutical compositions to treat neurological diseases or conditions, infections, or cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 484,440, filed February 10, 2023, the contents of which are incorporated herein by reference.

[0002]

[0002] The present invention specifically relates to a method for the isolation and preparation of exosome (NestaExo) compositions for therapeutic applications obtained from the supernatant of human immature dental pulp stem cells (hIDPSCs) from in vitro culture. [Background technology]

[0003]

[0003] Emergency cell-based therapies have offered many opportunities for the treatment of numerous incurable and critical illnesses, including neurodegenerative disorders (Araldi et al., 2020. Cells, 9, 12:2663), coronavirus disease 2019 (COVID-19) (Sadeghi et al., 2020. Life Science, 262:118493), and cancer (Hmadcha et al., 2020. Frontiers in Bioengineering and Biotechnology, 8, 43:1-13). In this regard, mesenchymal stem cells (MSCs) have been recognized as the mainstay of this novel therapeutic approach.

[0004] MSCs are multipotent cells that originate from a variety of adult, perinatal, and fetal tissues (Shamir et al., 2015. Neural Regenerative Research, Vol. 10, 12:1910-1911). However, it is not surprising that each type of MSC shares its origin and transcriptome signature, and therefore exhibits distinct therapeutic properties. In this regard, accumulating evidence has demonstrated that human immature dental pulp stem cells (hIDPSCs), a specialized type of adult MSC isolated from deciduous teeth of children between 6 and 12 years of age, are promising candidates for the treatment of neurodegenerative disorders (Araldi et al., 2020. Cells, Vol. 9, 12:2663). This is because, due to their ectomesenchymal origin (neural crest), hIDPSCs naturally produce and secrete numerous neural factors, including nestin (a neural stem / progenitor cell marker) and brain-derived neurotrophic factor (BDNF, which has been found to be downregulated in Huntington's disease-HD patients) (Wenceslau et al., 2022. Cells. Vol. 11(10), 1664 and Gonzaga et al., 2022. Cells. Vol. 11(914), 2252), yet they exhibit all the criteria for defining multipotent MSCs proposed by the International Society for Cellular Therapy (Dominici et al., 2006. Cytotherapy. Vol. 8(4), 315-317). For this reason, there has been significant interest in the clinical application of these cells for the treatment of neurodegenerative disorders, particularly neurodegenerative diseases (Venugopal et al., 2018. Current gene Therapy, vol. 18, 5:307-323 and Kerkis et al., 2022. InTech Open, 1-27).

[0005] For a long time, however, the therapeutic benefit of MSC-based therapies was thought to be associated with the replacement of dead cells. However, with the discovery that MSC-conditioned medium (CM), also known as the secretome, possesses regenerative potential similar to that of MSCs themselves, accumulating evidence supports the notion that part of MSC mechanism of action (MoA) is paracrinely mediated by bioactive molecules (coding and non-coding RNA, lipids, and metabolites) naturally produced by these cells and secreted either free or within extracellular vesicles (EVs) (Kumar et al., 2019, Cytokine & Growth Factor Reviews, Vol. 46, pp. 1–9). This discovery has led to a novel, innovative therapeutic proposal, which aims to use exosomes derived from MSC-conditioned medium for clinical purposes. This approach is known as cell-free therapy.

[0006] In this regard, it has been recognized that all cell types, including hIDPSCs, continuously produce and release small EVs, classified as microvesicles (100–1,000 nm), exosomes (30–150 nm), and apoptotic bodies (1,000–5,000 nm). These vesicles have been detected and isolated from various body fluids, such as blood, semen, urine, bile, ependymal fluid, amniotic fluid, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage fluid, pleural effusions of malignant and ascites, breast milk, saliva, and sweat. Given that these vesicles are naturally produced and released, they can also be isolated from cell culture supernatants.

[0007]

[0007] Among the various types of EVs, exosomes are the best characterized and widely used type of EVs, especially those for therapeutic purposes (Kalluri and LeBleu. 2020. Science, Vol. 367, 6478:eaau6977).

[0008]

[0008] Exosomes are a type of extracellular nanovesicle (typically approximately 30-200 nm) surrounded by a single membrane and express evolutionarily conserved biomarkers such as tetraspanins (CD9, CD63, CD81, and CD82), heat shock proteins (Hsp60, 70, and 90), major histocompatibility complex (MHC) class I and II, Alix, Tsg101, lactadherin, and lysosome-associated membrane glycoprotein 2. Interestingly, exosomes carry various bioactive molecules such as proteins, lipids, cytosolic components (mitochondria, endoplasmic reticulum, and Golgi apparatus), and nucleic acids, including coding RNA (messenger RNA - mRNA) and non-coding RNA (small RNA - sRNA, microRNA - miRNA, and long non-coding - lncRNA), and play an important role in intercellular communication associated with both physiological and pathological processes (Pegtel and Gould, 2019. Annual Reviews of Biochemistry, Vol. 88: 487-514).

[0009] Non-coding RNAs, such as miRNAs and lncRNAs, can regulate gene expression by targeting mRNA for degradation or blocking translation. These RNAs have been observed in many stem cell-derived exosomes and play an important role in intercellular communication (Chen, Lim 2013. Methods in Molecular Biology, Vol. 1024:69-86; Asgapour et al. 2020. Cell Communication and Signaling, Vol. 149:1-16).

[0010] Interestingly, recent studies suggest that specific motifs in miRNA sequences are recognized by RNAs bound to the sumoylated protein hnRNPA2B1, leading to selective incorporation of miRNAs into exosomes (Villarroya-Beltri et al., 2013. Nature Communications, Vol. 2890, pp. 1-10). This data demonstrates that, like MSCs, exosomes derived from various MSCs also possess unique transcriptome signatures and, therefore, distinct therapeutic properties (Araldi et al., 2020. Cells. Vol. 9(12), 2663).

[0011] In addition, the combined activity of multiple bioactive molecules present in exosomes can simultaneously act on various molecular pathways, including those associated with pathophysiological processes, those mediating cell regeneration functions, and those regulating inflammatory responses (Yin et al., 2019. Biomarker Research, Vol. 7, 8:1-8). For these reasons, exosomes have been explored in multiple preclinical trials for the treatment of several incurable diseases, including neurodegenerative disorders (Shaimardonova et al., 2020. Neural Regeneration Research, Vol. 15, 4:586-596).

[0012]

[0012] In this regard, preclinical studies have shown evidence that hIDPSCs are capable of improving motor, cognitive, and neuropsychiatric functions in the treatment of neurodegenerative disorders such as Huntington's disease (Wenceslau et al., 2022. Cells. Vol. 11(10), 1664) and Parkinson's disease (Araldi et al., 2022. Cytotherapy, Vol. 24(10), S3), and are safe and effective, suggesting that hIDPSC-derived exosomes could be used for clinical purposes (cell-free therapy) as an alternative treatment for these neurodegenerative disorders.

[0013] Neurodegenerative disorders affect millions of people worldwide and pose a major threat to human health. They are age-dependent disorders, and their prevalence is increasing, in part due to the recent increase in the aging population (Gilter et al., 2017. Disease Model Mechanism, Vol. 10, 5:499-502). The term "neurodegenerative disease" encapsulates a heterogeneous group of disorders, including Alzheimer's disease (AD), Parkinson's disease (PK), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and spinocerebellar ataxia. These diseases are characterized by abnormal protein aggregation, which leads to the inclusion of unfolded proteins in mitochondria, resulting in oxidative stress, neuroinflammation, and progressive neurodegeneration in the central nervous system (CNS) (Dugger and Dickson, 2017. Cold Spring Harbor Perspective Biology, Vol. 9, 7:a20835). While available treatments can help alleviate some of the physical and / or psychological symptoms, neurodegenerative diseases remain incurable. For this reason, novel therapies are essential to ameliorate the signs and symptoms of these diseases (Araldi et al., 2020. Cells, Vol. 9, 2663:1-29).

[0014]

[0014] Considering that accumulating evidence supports that congenital neuroinflammation mediated by microglia and astrocytes is a common denominator in neurodegenerative disorders (Dorothee, 2018. Journal of Neuronal Transmission, Vol. 125:749-750), it is not surprising that therapeutic agents capable of ameliorating the signs and symptoms of neurodegenerative disorders could also be used to treat other diseases whose pathophysiology is based on inflammation, such as COVID-19 and cancer.

[0015] COVID-19 is an emerging disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Huang et al., 2020. The Lancet, Vol. 395, 10223:497-506). Since the first case of the disease was reported in Wuhan, China, in December 2019, over 124 million people have been infected with SARS-CoV-2, and 2.7 million people worldwide have died from COVID-19-related complications. The majority of these deaths are associated with cytokine storm syndrome, a term commonly used to describe the uncontrolled overexpression of proinflammatory cytokines and chemokines (Coperchini et al., 2020. Cytokine & Growth Factor Reviews, Vol. 53:25-32). Despite efforts to develop promising therapies to combat COVID-19, no approved effective treatments have been found to date that can halt the progression of COVID-19 or address severe cases with high mortality rates, sparking social unrest during the "coronavirus pandemic" (Pamuru et al., 2020. Current Pharmaceutical Design, Vol. 26, 41:5278-5285). Therefore, there is a strong need for treatments that can mitigate severe COVID-19, thereby reducing case fatality rates and speeding the recovery of critically ill patients. In this regard, the International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recently recognized the potential of MSC-derived extracellular vesicles, including exosomes, as a treatment for COVID-19 (Borger et al., 2020. Cytotherapy, Vol. 22, 9:482-485). This is because the use of these exosomes not only reduces the cytokine storm through immunomodulatory mechanisms but also exerts a regenerative effect (Rezakhani et al., 2020. Chemistry and Physics of Lipids, Vol. 234, 105009, 1-6).

[0016] Despite advances in oncology, cancer remains a major public health problem and the second leading cause of death worldwide. Statistics indicate that an estimated 608,570 Americans will die from cancer in 2021, representing more than 1,600 deaths per day (Siegel et al., 2021. CA: A Cancer Journal for Clinicians, Vol. 71, pp. 177-178). Metastasis is the leading cause of cancer death, accounting for more than 90% of total cancer deaths (Dillekas et al., 2019. Cancer Medicine, Vol. 48, pp. 125-155). Current research indicates that inflammation not only predisposes to cancer development but also promotes all stages of carcinogenesis (Greten et al., 2019. Immunity, Vol. 51, pp. 125-157). Emerging evidence supports that MSC-derived exosomes promote immune regulation of the tumor microenvironment in an interleukin-6 (IL-6)-dependent manner, resulting in antitumor effects (Xunian, Kalluri. 2020. Cancer Science, Vol. 111: 3100-3110).

[0017]

[0017] Despite evidence of the therapeutic potential of exosomes, cell-free based therapies require large amounts of exosomes, which limits the use of this technology for clinical applications.

[0018]

[0018] Various strategies for isolating exosomes, such as differential or density gradient ultracentrifugation (UC), size-exclusion or affinity chromatography, immunoaffinity capture-based techniques, sequential filtration, and tangential flow filtration (TFF), have been successfully used, but none of them are scalable.

[0019]

[0019] Therefore, the present invention provides a novel, scalable method for generating and preparing exosomes (NestaExo) derived from the conditioned culture medium of human immature dental pulp stem cells (hIDPSCs) for therapeutic use. Summary of the Invention [Means for solving the problem]

[0020]

[0020] In one embodiment, the present invention provides a method for isolating exosomes from a culture of cells, the method comprising the steps of obtaining a cell culture medium from the culture of cells, removing cells and cell debris from the cell culture medium without centrifuging the cell culture medium, and subjecting the cell- and cell-debris-free cell culture medium to ultracentrifugation at 3-6°C to produce a pellet containing exosomes. In some embodiments, the step of removing cells and cell debris comprises filtering the cell culture medium to obtain a filtrate, which is subjected to ultracentrifugation. In certain implementations, the step of removing cells and cell debris from the cell culture medium comprises filtering the cell culture medium to obtain a filtrate, which is subjected to ultracentrifugation. In some embodiments, the cell culture medium is filtered using a 0.22 μm cellulose acetate membrane. In certain implementations, the filtrate is subjected to ultracentrifugation for at least 40 minutes, such as between 40 and 70 minutes, at a speed of 99,850 to 100,210 × g (RCF) or 99,650 to 100,350 × g (RCF). In certain implementations, the filtrate is subjected to ultracentrifugation at a speed of 100,000 × g (RCF). In such implementations, the filtrate is subjected to ultracentrifugation for 60 minutes, preferably at 4°C. In certain embodiments, ultracentrifugation is performed in a centrifuge equipped with a swing-out rotor that holds centrifuge tubes with a capacity of at least 15 mL.

[0021] The resulting pellet containing exosomes does not contain polyethylene glycol (PEG) because PEG was not used to isolate the exosomes. In preparing a therapeutic composition containing isolated exosomes, the method further comprises resuspending the pellet in saline or other solution. In some embodiments, the saline solution is 0.9% sterile saline. In other embodiments, the saline solution is isotonic. Thus, in some embodiments, pharmaceutical compositions comprising isolated exosomes and a pharmaceutically acceptable carrier or excipient are described. In some embodiments, the pharmaceutical composition comprises isolated exosomes resuspended in isotonic saline. In one embodiment, the pharmaceutical composition is formulated for local, intranasal, intravenous (IV), or intrathecal injection.

[0022] In certain implementations, the method further comprises culturing the cells in serum-free medium for at least 12 hours and then recovering the serum-free medium after the cells have been cultured in serum-free medium for at least 12 hours, thereby obtaining cell culture medium from the cell culture. In some embodiments, the serum-free medium is recovered after the cells have been cultured in serum-free medium for 48 hours or less.

[0023]

[0023] In some embodiments, a method for isolating exosomes from a culture of cells consists essentially of obtaining cell culture medium from the cell culture, filtering the cell culture medium to obtain a filtrate, thereby removing cells and cell debris from the cell culture medium, subjecting the filtrate to ultracentrifugation at 3-6°C to produce a pellet containing exosomes, and optionally resuspending the pellet.

[0024]

[0024] Also described is a method for isolating exosomes from human immature dental pulp stem cells (hIDPSCs). The method includes culturing hIDPSCs in a basal medium, replacing the basal medium with serum-free medium when the hIDPSCs reach at least 80% confluence, and recovering the serum-free medium after the hIDPSCs have been cultured in the serum-free medium for at least 12 hours. In some implementations, the serum-free medium is recovered after the hIDPSCs have been cultured in the serum-free medium for 48 hours or less. In some embodiments, the basal medium is replaced with serum-free medium when the hIDPSCs do not reach greater than 90% confluence. The method further includes removing cells and cell debris from the recovered serum-free medium, including filtering the recovered serum-free medium to obtain a filtrate, but not centrifuging the recovered serum-free medium; ultracentrifuging the filtrate at 3-6°C to produce a pellet containing exosomes; and, optionally, resuspending the pellet. The pellet may be resuspended in saline, e.g., 0.9% sterile saline and / or isotonic saline. The resulting pellet containing exosomes is PEG-free, since no PEG was used to isolate the exosomes.

[0025]

[0025] In certain implementations, removing cells and cell debris from the cell culture medium comprises filtering the cell culture medium to obtain a filtrate, and the filtrate is subjected to ultracentrifugation. In some embodiments, the cell culture medium is filtered using a 0.22 μm cellulose acetate membrane. In certain implementations, the filtrate is subjected to ultracentrifugation at a speed of 99,850 to 100,210 × g (RCF) or 99,650 to 100,350 × g (RCF) for at least 40 minutes, such as between 40 and 70 minutes. In certain implementations, the filtrate is subjected to ultracentrifugation at a speed of 100,000 × g (RCF). In such implementations, the filtrate is subjected to ultracentrifugation for 60 minutes, preferably at 4°C. In certain embodiments, ultracentrifugation is performed in a centrifuge equipped with a swing rotor that holds centrifuge tubes with a capacity of at least 15 mL.

[0026]

[0026] In some embodiments, the serum-free medium comprises Dulbecco's Modified Eagle's Medium (DMEM) / Ham's F12 (DMEM / F12) supplemented with 100 units / mL penicillin, 100 μg / mL streptomycin, 2 nM L-glutamine, and 2 mM non-essential amino acids.

[0027] The exosomes isolated from the above method have a diameter of 20 to 180 nm, e.g., 40 to 120 nm. In yet another embodiment, the exosomes have a diameter of 20 to 60 nm. In some embodiments, at least 1 x 10 exosomes are isolated per mL of filtrate. 10 Exosomes are isolated.

[0028] In some embodiments, the isolated exosomes express at least one marker selected from integrin, intercellular adhesion molecule 1 (ICAM-1), epithelial cell adhesion molecule (EpCAM), CD31, annexin, TSG01, apoptosis-related gene-interacting protein X (ALIX), Rab5b, HLA-G, HSP70, CD63, lysosomal-associated membrane protein 2 (LAMP2), and lysosomal integral membrane protein (LIMP). In certain embodiments, the at least one marker is CD63. In certain embodiments, exosomes isolated from hIDPSCs according to the methods described herein express 5,817 transcripts, including 74 gene products that are uniquely expressed by hIDPSCs but not identified in other MSCs. The transcripts contained within exosomes confer neuroprotective and neuroregenerative potential.

[0029] In another aspect, the isolated exosomes comprise proteins, lipids, cytosolic components, and nucleic acids. In a specific embodiment, the nucleic acids comprise miRNAs and lncRNAs.

[0030]

[0030] The present invention further provides a method of treating a neurological disease or condition, an infectious disease, or cancer, comprising administering exosomes isolated using the above-described methods to a subject, such as via the disclosed pharmaceutical composition. In certain embodiments, the pharmaceutical composition is administered intravenously to the subject.

[0031] In another embodiment, the present invention provides a pharmaceutical composition comprising exosomes for use in the treatment of a neurodegenerative disorder or condition, an infectious disease (particularly COVID-19), or cancer. In a specific embodiment, the neurological disease or condition is ALS, HD, or PD. In yet another embodiment, the infectious disease is novel coronavirus disease (COVID-19).

[0032]

[0032] These and other aspects of the present invention will become apparent in the detailed description that follows and by reference to the following figures.

[0033] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0033] [Figure 1]

[0034] Figure 1A shows hIDPSCs cultured in complete medium (i.e., containing fetal serum). Figure 1B shows that 24 hours of fetal serum starvation (required for isolating exosomes from culture medium) did not promote any morphological changes in hIDPSCs; hIDPSCs remained attached to plastic and exhibited a fibroblast-like morphology similar to hIDPSCs cultured in complete medium (i.e., containing fetal serum), suggesting that 24 hours of serum starvation did not promote any morphological changes in hIDPSCs. [Figure 2A]

[0035] Even after 24 hours of serum starvation, hIDPSCs maintained MSC marker expression and were immunonegative for CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), in accordance with the minimal criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 2B] Even after 24 hours of serum starvation, hIDPSCs maintained MSC marker expression and were immunonegative for CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), in accordance with the minimal criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 2C] Even after 24 hours of serum starvation, hIDPSCs maintained MSC marker expression and were immunonegative for CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), in accordance with the minimal criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 2D] Even after 24 hours of serum starvation, hIDPSCs maintained MSC marker expression and were immunonegative for CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), in accordance with the minimal criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 2E]Even after 24 hours of serum starvation, hIDPSCs maintained MSC marker expression and were immunonegative for CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), in accordance with the minimal criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 2F] Even after 24 hours of serum starvation, hIDPSCs maintained MSC marker expression and were immunonegative for CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), in accordance with the minimal criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 2G] Even after 24 hours of serum starvation, hIDPSCs maintained MSC marker expression and were immunonegative for CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), in accordance with the minimal criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 3A]

[0036] 3A-3D , hIDPSCs were immunopositive for CD73 (FIG. 3A), CD90 (FIG. 3B), CD105 (FIG. 3C), and CD146 (FIG. 3D) after 24 hours of serum starvation, demonstrating that these cells maintained positive expression of these cellular markers according to the criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 3B]3A-3D , hIDPSCs were immunopositive for CD73 (FIG. 3A), CD90 (FIG. 3B), CD105 (FIG. 3C), and CD146 (FIG. 3D) after 24 hours of serum starvation, demonstrating that these cells maintained positive expression of these cellular markers according to the criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 3C] 3A-3D , hIDPSCs were immunopositive for CD73 (FIG. 3A), CD90 (FIG. 3B), CD105 (FIG. 3C), and CD146 (FIG. 3D) after 24 hours of serum starvation, demonstrating that these cells maintained positive expression of these cellular markers according to the criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 3D] 3A-3D , hIDPSCs were immunopositive for CD73 (FIG. 3A), CD90 (FIG. 3B), CD105 (FIG. 3C), and CD146 (FIG. 3D) after 24 hours of serum starvation, demonstrating that these cells maintained positive expression of these cellular markers according to the criteria for defining multipotent mesenchymal stem cells proposed by the International Society for Cellular Therapy. [Figure 4A]

[0037] Figure 4A shows transmission electron microscopy analysis of extracellular vesicles isolated using 1) the method disclosed herein (Figure 4A) or 2) a PEG-based commercial kit (Figure 4B) and an immunoaffinity capture-based protocol using a commercial kit with immunodetection of CD63 (Figure 4C). The results demonstrate that the method disclosed herein can isolate pure extracellular vesicles without vesicle aggregates in residual PEG (Figure 4A). In contrast, the presence of vesicle aggregates in residual PEG was observed with both commercial methods (Figures 4B and 4C). [Figure 4B]Figure 4A shows transmission electron microscopy analysis of extracellular vesicles isolated using 1) the method disclosed herein (Figure 4A) or 2) a PEG-based commercial kit (Figure 4B) and an immunoaffinity capture-based protocol using a commercial kit with immunodetection of CD63 (Figure 4C). The results demonstrate that the method disclosed herein can isolate pure extracellular vesicles without vesicle aggregates in residual PEG (Figure 4A). In contrast, the presence of vesicle aggregates in residual PEG was observed with both commercial methods (Figures 4B and 4C). [Figure 4C] Figure 4A shows transmission electron microscopy analysis of extracellular vesicles isolated using 1) the method disclosed herein (Figure 4A) or 2) a PEG-based commercial kit (Figure 4B) and an immunoaffinity capture-based protocol using a commercial kit with immunodetection of CD63 (Figure 4C). The results demonstrate that the method disclosed herein can isolate pure extracellular vesicles without vesicle aggregates in residual PEG (Figure 4A). In contrast, the presence of vesicle aggregates in residual PEG was observed with both commercial methods (Figures 4B and 4C). [Figure 5A]

[0038] FIG. 5A shows atomic force microscopy (AFM) confirming the purity of extracellular vesicles isolated using the disclosed methods. Furthermore, AFM revealed the presence of clumps of PEG precipitate (i.e., impurities) in extracellular vesicles isolated using both commercially available methods: a PEG-based co-precipitation method (FIG. 5B) and an immunoaffinity capture-based method (FIG. 5C). [Figure 5B] FIG. 5A shows atomic force microscopy (AFM) confirming the purity of extracellular vesicles isolated using the disclosed methods. Furthermore, AFM revealed the presence of clumps of PEG precipitate (i.e., impurities) in extracellular vesicles isolated using both commercially available methods: a PEG-based co-precipitation method (FIG. 5B) and an immunoaffinity capture-based method (FIG. 5C). [Figure 5C]FIG. 5A shows atomic force microscopy (AFM) confirming the purity of extracellular vesicles isolated using the disclosed methods. Furthermore, AFM revealed the presence of clumps of PEG precipitate (i.e., impurities) in extracellular vesicles isolated using both commercially available methods: a PEG-based co-precipitation method (FIG. 5B) and an immunoaffinity capture-based method (FIG. 5C). [Figure 6A]

[0039] Figure 6A shows AFM performed in liquid medium confirming the purity of extracellular vesicles isolated using the disclosed method (Figure 6A) and the presence of PEG precipitate clumps (i.e., impurities) in extracellular vesicles isolated using a commercially available method (Figures 6B and 6C). [Figure 6B] Figure 6A shows AFM performed in liquid medium confirming the purity of extracellular vesicles isolated using the disclosed method (Figure 6A) and the presence of PEG precipitate clumps (i.e., impurities) in extracellular vesicles isolated using a commercially available method (Figures 6B and 6C). [Figure 6C] Figure 6A shows AFM performed in liquid medium confirming the purity of extracellular vesicles isolated using the disclosed method (Figure 6A) and the presence of PEG precipitate clumps (i.e., impurities) in extracellular vesicles isolated using a commercially available method (Figures 6B and 6C). [Figure 7A]

[0040] Figure 7A shows the absence of PEG precipitate clumps in extracellular vesicles isolated using the disclosed method, and Figure 7B shows the presence or absence of PEG contamination in extracellular vesicles isolated using a PEG-based co-precipitation method, obtained using atomic force microscopy (AFM) 3D analysis. [Figure 7B] Figure 7A shows the absence of PEG precipitate clumps in extracellular vesicles isolated using the disclosed method, and Figure 7B shows the presence or absence of PEG contamination in extracellular vesicles isolated using a PEG-based co-precipitation method, obtained using atomic force microscopy (AFM) 3D analysis. [Figure 8A]

[0041] Figure 8A shows the results of nanoparticle tracking analysis (NTA) of extracellular vesicles isolated using the disclosed method. Six ultracentrifuge tubes containing 13 mL of conditioned culture medium were subjected to ultracentrifugation for 1 hour each, according to the described method. At the end of ultracentrifugation, after discarding the supernatant (volume of approximately 12.8 mL), a final volume of approximately 0.2 mL containing exosomes was obtained. Approximately 10 μL of the exosome fraction was taken to measure exosome concentration using NTA. Figure 8A shows vesicles identified by the NanoSight NS300 nanoparticle tracking analyzer. Five consecutive analyses showed that vesicles isolated using the disclosed method had a moderate diameter of 100.7 ± 1.2 nm, which is consistent with the expected diameter of exosomes (30–150 nm) (Figure 8B). The results demonstrate that the disclosed method not only enables the isolation of large amounts of exosomes (i.e., approximately 1.12 x 10 exosomes / mL or approximately 0.86 x 10 exosomes / mL in serum-free medium) (Figure 8B), but also ensures homogeneity of the isolated exosomes, which can be evidenced by limited polydispersity (Figure 8C). [Figure 8B]Figure 8A shows the results of nanoparticle tracking analysis (NTA) of extracellular vesicles isolated using the disclosed method. Six ultracentrifuge tubes containing 13 mL of conditioned culture medium were subjected to ultracentrifugation for 1 hour each, according to the described method. At the end of ultracentrifugation, after discarding the supernatant (volume of approximately 12.8 mL), a final volume of approximately 0.2 mL containing exosomes was obtained. Approximately 10 μL of the exosome fraction was taken to measure exosome concentration using NTA. Figure 8A shows vesicles identified by the NanoSight NS300 nanoparticle tracking analyzer. Five consecutive analyses showed that vesicles isolated using the disclosed method had a moderate diameter of 100.7 ± 1.2 nm, which is consistent with the expected diameter of exosomes (30–150 nm) (Figure 8B). The results demonstrate that the disclosed method not only enables the isolation of large amounts of exosomes (i.e., approximately 1.12 x 10 exosomes / mL or approximately 0.86 x 10 exosomes / mL in serum-free medium) (Figure 8B), but also ensures homogeneity of the isolated exosomes, which can be evidenced by limited polydispersity (Figure 8C). [Figure 8C]Figure 8A shows the results of nanoparticle tracking analysis (NTA) of extracellular vesicles isolated using the disclosed method. Six ultracentrifuge tubes containing 13 mL of conditioned culture medium were subjected to ultracentrifugation for 1 hour each, according to the described method. At the end of ultracentrifugation, after discarding the supernatant (volume of approximately 12.8 mL), a final volume of approximately 0.2 mL containing exosomes was obtained. Approximately 10 μL of the exosome fraction was taken to measure exosome concentration using NTA. Figure 8A shows vesicles identified by the NanoSight NS300 nanoparticle tracking analyzer. Five consecutive analyses showed that vesicles isolated using the disclosed method had a moderate diameter of 100.7 ± 1.2 nm, which is consistent with the expected diameter of exosomes (30–150 nm) (Figure 8B). The results demonstrate that the disclosed method not only enables the isolation of large amounts of exosomes (i.e., approximately 1.12 x 10 exosomes / mL or approximately 0.86 x 10 exosomes / mL in serum-free medium) (Figure 8B), but also ensures homogeneity of the isolated exosomes, which can be evidenced by limited polydispersity (Figure 8C). [Figure 9A]

[0042] Figure 9 shows immunodetection of exosome marker CD63 by imaging flow cytometry. The results show immunolabeling of exosome marker CD63 on the surface of extracellular vesicles isolated using the methods disclosed herein (Figure 9A). In this assay, 20,000 events were analyzed, and 98.9% of the isolated vesicles were confirmed to be CD63 positive, demonstrating that the disclosed methods enable the isolation of exosomes with high purity (Figure 9B). [Figure 9B]Figure 9 shows immunodetection of exosome marker CD63 by imaging flow cytometry. The results show immunolabeling of exosome marker CD63 on the surface of extracellular vesicles isolated using the methods disclosed herein (Figure 9A). In this assay, 20,000 events were analyzed, and 98.9% of the isolated vesicles were confirmed to be CD63 positive, demonstrating that the disclosed methods enable the isolation of exosomes with high purity (Figure 9B). [Figure 10A]

[0043] Figures 10A and 10B show the results of exosome stability after 90 days of storage at 4°C (Figures 10A and 10B), -20°C (Figures 10C and 10D), and -80°C (Figures 10E and 10F). The results indicate that temperature and storage period (i.e., 90 days) do not affect exosome size, with mean diameters of 93.1 nm at 4°C (Figure 10A), 98.0 nm at -20°C (Figure 10C), and 94.3 nm at -80°C (Figure 10E). However, storage at 4°C resulted in a decreased number of exosomes (7.16×109 particles / mL, Figures 10A and 10B) compared to storage at -20°C (1.28×1011 particles / mL, Figures 10C and 10D) and -80°C (1.43×1011 particles / mL, Figures 10E and 10F), suggesting that storage at -20°C and -80°C temperatures improves the preservation of hIDPSC-derived exosomes. [Figure 10B]Figures 10A and 10B show the results of exosome stability after 90 days of storage at 4°C (Figures 10A and 10B), -20°C (Figures 10C and 10D), and -80°C (Figures 10E and 10F). The results indicate that temperature and storage period (i.e., 90 days) do not affect exosome size, with mean diameters of 93.1 nm at 4°C (Figure 10A), 98.0 nm at -20°C (Figure 10C), and 94.3 nm at -80°C (Figure 10E). However, storage at 4°C resulted in a decreased number of exosomes (7.16×109 particles / mL, Figures 10A and 10B) compared to storage at -20°C (1.28×1011 particles / mL, Figures 10C and 10D) and -80°C (1.43×1011 particles / mL, Figures 10E and 10F), suggesting that storage at -20°C and -80°C temperatures improves the preservation of hIDPSC-derived exosomes. [Figure 10C] Figures 10A and 10B show the results of exosome stability after 90 days of storage at 4°C (Figures 10A and 10B), -20°C (Figures 10C and 10D), and -80°C (Figures 10E and 10F). The results indicate that temperature and storage period (i.e., 90 days) do not affect exosome size, with mean diameters of 93.1 nm at 4°C (Figure 10A), 98.0 nm at -20°C (Figure 10C), and 94.3 nm at -80°C (Figure 10E). However, storage at 4°C resulted in a decreased number of exosomes (7.16×109 particles / mL, Figures 10A and 10B) compared to storage at -20°C (1.28×1011 particles / mL, Figures 10C and 10D) and -80°C (1.43×1011 particles / mL, Figures 10E and 10F), suggesting that storage at -20°C and -80°C temperatures improves the preservation of hIDPSC-derived exosomes. [Figure 10D]Figures 10A and 10B show the results of exosome stability after 90 days of storage at 4°C (Figures 10A and 10B), -20°C (Figures 10C and 10D), and -80°C (Figures 10E and 10F). The results indicate that temperature and storage period (i.e., 90 days) do not affect exosome size, with mean diameters of 93.1 nm at 4°C (Figure 10A), 98.0 nm at -20°C (Figure 10C), and 94.3 nm at -80°C (Figure 10E). However, storage at 4°C resulted in a decreased number of exosomes (7.16×109 particles / mL, Figures 10A and 10B) compared to storage at -20°C (1.28×1011 particles / mL, Figures 10C and 10D) and -80°C (1.43×1011 particles / mL, Figures 10E and 10F), suggesting that storage at -20°C and -80°C temperatures improves the preservation of hIDPSC-derived exosomes. [Figure 10E] Figures 10A and 10B show the results of exosome stability after 90 days of storage at 4°C (Figures 10A and 10B), -20°C (Figures 10C and 10D), and -80°C (Figures 10E and 10F). The results indicate that temperature and storage period (i.e., 90 days) do not affect exosome size, with mean diameters of 93.1 nm at 4°C (Figure 10A), 98.0 nm at -20°C (Figure 10C), and 94.3 nm at -80°C (Figure 10E). However, storage at 4°C resulted in a decreased number of exosomes (7.16×109 particles / mL, Figures 10A and 10B) compared to storage at -20°C (1.28×1011 particles / mL, Figures 10C and 10D) and -80°C (1.43×1011 particles / mL, Figures 10E and 10F), suggesting that storage at -20°C and -80°C temperatures improves the preservation of hIDPSC-derived exosomes. [Figure 10F]Figures 10A and 10B show the results of exosome stability after 90 days of storage at 4°C (Figures 10A and 10B), -20°C (Figures 10C and 10D), and -80°C (Figures 10E and 10F). The results indicate that temperature and storage period (i.e., 90 days) do not affect exosome size, with mean diameters of 93.1 nm at 4°C (Figure 10A), 98.0 nm at -20°C (Figure 10C), and 94.3 nm at -80°C (Figure 10E). However, storage at 4°C resulted in a decreased number of exosomes (7.16×109 particles / mL, Figures 10A and 10B) compared to storage at -20°C (1.28×1011 particles / mL, Figures 10C and 10D) and -80°C (1.43×1011 particles / mL, Figures 10E and 10F), suggesting that storage at -20°C and -80°C temperatures improves the preservation of hIDPSC-derived exosomes. [Figure 11]

[0044] Figure 1 shows electropherograms of 50 μg of total protein isolated from freshly isolated exosomes derived from hIDPSCs of two different donors and from exosomes stored for 90 days at -20°C. The results show very similar electropherograms, demonstrating that long-term storage does not promote changes in protein content. [Figure 12A]

[0045] Figure 12A shows the integrity and concentration of total RNA isolated from exosomes freshly derived from hIDPSCs (Figure 12A) and exosomes stored at -20°C for 90 days (Figure 12B), both obtained using the methods of the invention. The results demonstrate that long-term storage at -20°C did not compromise RNA integrity or concentration. [Figure 12B] Figure 12A shows the integrity and concentration of total RNA isolated from exosomes freshly derived from hIDPSCs (Figure 12A) and exosomes stored at -20°C for 90 days (Figure 12B), both obtained using the methods of the invention. The results demonstrate that long-term storage at -20°C did not compromise RNA integrity or concentration. [Figure 13A]

[0046] Figure 13A shows a graphical representation of the average quality control scores for four preparations of hIDPSCs (Figure 13A) and the exosomes secreted from each of these preparations (Figure 13B) after analysis using RNA-Sep. The results demonstrate high quality (Phred score >36) of all raw data after Illumina adapter trimming. [Figure 13B] Figure 13A shows a graphical representation of the average quality control scores for four preparations of hIDPSCs (Figure 13A) and the exosomes secreted from each of these preparations (Figure 13B) after analysis using RNA-Sep. The results demonstrate high quality (Phred score >36) of all raw data after Illumina adapter trimming. [Figure 14A]

[0047] Figure 14 shows QualiMap results demonstrating the genomic origin of the reads, revealing that 70% of the aligned reads from the hIDPSC sample (Figure 14A) and 56% of the aligned reads from the exosome sample (Figure 14B) were within exonic regions. [Figure 14B] Figure 14 shows QualiMap results demonstrating the genomic origin of the reads, revealing that 70% of the aligned reads from the hIDPSC sample (Figure 14A) and 56% of the aligned reads from the exosome sample (Figure 14B) were within exonic regions. [Figure 15]

[0048] A Venn diagram of the mRNA transcripts identified in hIDPSCs (NestaCell® product) and four different batches of NestaExo shows that NestaExo has 6,580 different mRNAs, representing 80.96% of the transcripts expressed by the active components of the NestaCell® product (hIDPSCs, 6,580 / 8,127). [Figure 16]

[0049] Figure 11 graphically depicts the comparative transcriptomes of hIDPSCs (NestaCell® product) and hIDPSC-derived exosomes (NestaExo) using different brain regions. The results reveal that the two batches of NestaCell® and NestaExo share over 50% of the transcripts expressed in the hippocampus, cerebellum, cerebral cortex, and lateral ventricles, suggesting that either product could be used as a therapeutic agent for neurodegenerative disorders. [Figure 17]

[0050] Figure 1 shows transcription factors identified at high levels in both hIDPSCs (NestaCell® product) and hIDPSC-derived exosomes (NestaExo). These transcription factors (SP1, Klf4, SP4, EGR1, NFIC) can promote neural stem cell proliferation and differentiation into neurons, driving neurogenesis. [Figure 18]

[0051] This figure shows the results of KEGG term enrichment analysis. The analysis was performed based on a list of coding RNA (mRNA) transcripts commonly identified using exosomes from two hIDPSC donors and a list of coding RNA (mRNA) transcripts exclusively identified as DE (i.e., differentially expressed) in exosomes. The results indicate that mRNAs delivered by NestaExo can regulate adherens junctions, which are disrupted in all malignancies, in addition to pathways related to amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). These data demonstrate the potential use of NestaExo exosomes as therapeutic agents for the prevention or treatment of neurodegenerative disorders, including but not limited to ALS and HD, as well as cancer. [Figure 19]

[0052] This figure shows the results of KEGG term enrichment analysis. The analysis was performed based on a list of lncRNA transcripts of non-coding RNAs commonly identified using exosomes from two hIDPSC donors and a list of lncRNA transcripts of non-coding RNAs exclusively identified as DE (i.e., differentially expressed) in exosomes. The results indicate that lncRNAs delivered by NestaExo exosomes can regulate several pathways recognized to be deregulated in cancer, including focal adhesions and extracellular matrix (ECM)-receptor interactions (deregulation of which is closely related to cancer metastasis) and proteoglycans (related to immune evasion) in cancer, in addition to genes deregulated in amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). These results highlight the potential use of NestaExo exosomes for the prevention and treatment of neurodegenerative disorders and cancer, including, but not limited to, ALS and HD. [Figure 20]

[0053] This figure shows the results of KEGG term enrichment analysis. The analysis was performed based on a list of microRNA transcripts of non-coding RNAs commonly identified using exosomes from two hIDPSC donors and a list of microRNA transcripts of non-coding RNAs exclusively identified as DE (i.e., differentially expressed) in exosomes. The results indicate that microRNAs delivered by NestaExo exosomes can regulate multiple pathways found to be deregulated in cancer, including the hypoxia-inducible factor 1 (HIF-1) signaling pathway, which acts as a driver of metabolic reprogramming associated with enrichment of pathways related to glycolysis and central carbon metabolism in cancer. These results highlight the potential for NestaExo exosomes to be used for the prevention or treatment of neurodegenerative disorders, COVID-19, and cancer. [Figure 21]

[0054] FIG. 10 graphically depicts the reduction of the neuroblastoma-derived cancer cell line SH-SY5Y following 48 hours of treatment with 50 μg of hIDPSC-derived exosomes (NestaExo). [Figure 22]

[0055] FIG. 1 shows protein array results demonstrating that treatment with hIDPSC-derived exosomes (NestaExo) was able to downregulate the expression levels of 12 cancer-associated proteins after 48 hours of treatment, 9 of which were dose-dependent. [Figure 23]

[0056] 1 shows the results of apomorphine challenge demonstrating neurological damage caused by intrastriatal injection of 6-hydroxydopamine (6-OHDA). Neurological damage was confirmed by the increased number of rotations demonstrated in rats treated with 6-OHDA (test group, placebo intravenous (IV) and intranasal (IN) and NestaCell-IV and IN). [Figure 24]

[0057] This figure shows the results of an open-field behavioral analysis confirming that intrastriatal injection of 6-OHDA caused neuronal damage to the substantia nigra. This shows that rats treated with saline (placebo) via the intravenous (IV) and intranasal (IN) routes exhibited reduced motor function compared with rats subjected to the intrastriatal procedure but not treated with 6-OHDA (Sham-control group). Meanwhile, rats subjected to intrastriatal injection of 6-OHDA but treated with NestaExo via the IV and IN routes showed improved motor function only 73 hours after product administration (Day 3 - D3). Interestingly, this improvement persisted for the 30-day analysis (D30) after IV administration. However, the motor effects of NestaExo declined after 15 days (D15) of product administration, suggesting that weekly administration via the IN route is required to maintain the benefits of the product. [Figure 25]

[0058] 1 shows the time that rats stayed in the center of the open field, which is used as a behavioral parameter for analyzing anxiety levels. The results show that rats that received intrastriatal injection of 6-OHDA and were treated with saline via intravenous (IV) or intranasal (IN) route (placebo-IV or IN) spent less time in the center of the arena compared with rats that were subjected to intrastriatal manipulation but not treated with 6-OHDA (Sham-control group), suggesting that 6-OHDA induced behavioral changes and increased the anxiety levels of the rats. Meanwhile, rats that were treated with NestaExo via IV or IN route spent more time in the center of the arena, suggesting that NestaExo has anxiolytic activity after 3 days (D3) of administration. The anxiolytic effect of NestaExo was observed for 15 days of product administration. [Figure 26]

[0059] This figure shows the grooming time recorded during the open field test, which was used as an additional parameter for analyzing the animal's anxiety level. The results show that rats receiving intrastriatal injections of 6-OHDA and treated with saline via intravenous (IV) or intranasal (IN) routes (placebo-IV or IN) showed a decrease in grooming time compared with rats subjected to the intrastriatal procedure but not treated with 6-OHDA (sham-control group). This result clearly demonstrates that intrastriatal injection of 6-OHDA increases anxiety levels (a behavior commonly observed in Parkinson's disease patients). On the other hand, rats treated with NestaExo via IV or IN routes showed an increase in grooming time to a statistically equivalent level to the sham group after 3 days (D3) of administration, demonstrating that NestaExo has an anxiolytic effect for at least 30 days (D30). DETAILED DESCRIPTION OF THE INVENTION

[0034]

[0001] Detailed aspects and applications of the present invention are described in the following drawings and detailed description. Unless otherwise specified, it is intended that the words and phrases in the specification and claims be given their obvious, ordinary, and accustomed meanings to those of ordinary skill in the relevant art.

[0035]

[0002] In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various aspects of the present invention. However, it will be understood by those skilled in the relevant art that the present invention may be practiced without these specific details. It should be noted that there are many different and alternative structures, devices, and techniques to which the disclosed invention may be applied. The full scope of the present invention is not limited to the examples set forth below.

[0036]

[0003] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "step" includes a reference to one or more of such steps.

[0037]

[0060] The term "exosomes" refers to small extracellular vesicles approximately 30-150 nm in diameter that express the surface biomarker CD63, which is not present on other vesicles. Exosomes also contain adhesion markers (integrins, ICAM-1, EpCAM, and CD31), membrane fusion markers (annexins, TSG01, and ALIX), and other exosomal transmembrane proteins (Rab5b, HLA-G, HSP70, lysosomal-associated membrane protein-LAMP2, and lysosomal integral membrane protein-LIMP). Exosomes can be isolated from any suitable biological sample, including body fluids and cultured mammalian cells such as hIDPSCs. However, to increase the number of free exosomes, the cell culture must be maintained in serum-free culture medium for at least 12 hours.

[0038]

[0061] "Human immature dental pulp stem cells," also referred to herein as "hIDPSCs," are a subpopulation of cells isolated from the dental pulp of deciduous teeth that meet the minimal criteria for multipotent mesenchymal stem cells defined by the International Society for Cellular Therapy (Dominici et al., 2006. Cytotherapy 8:4:315-317) and are applicable to cell-based therapies for a variety of diseases, including neurodegenerative disorders (Raza et al., 2018. Stem Cell Research Therapy 9:245:1-15; U.S. Patent No. 9,790,468 B2). The hIDPSCs utilized in the present methods are generally generated according to the methods disclosed in U.S. Patent No. 9,790,468, the contents of which are incorporated herein by reference in their entirety.

[0039]

[0062] As used herein, "NestaExo" refers to exosomes isolated from hIDPSCs.

[0063] As used herein, the term "treatment" or "treating" refers to any treatment of a target condition (e.g., cancer), including, but not limited to, prophylactic and therapeutic treatment. Thus, the term "treatment" or "treating" includes, but is not limited to, preventing the target condition or the occurrence of the target condition, inhibiting the progression of the target condition, arresting or preventing further occurrence of the target condition, reducing the severity of the target condition, ameliorating or alleviating the symptoms associated with the target condition, and reversing the target condition or one or more symptoms associated with the target condition.

[0040]

[0064] A novel method for isolating exosomes from cell culture supernatant (the clear liquid above the settled sediment) is provided. Prior art centrifugation-based methods for isolating exosomes involve two steps: a first removal step to remove cells and cell debris from the cell culture supernatant, and a second isolation step to isolate exosomes from the cleared cell culture supernatant. The removal step typically involves centrifugation and / or filtration of the cell culture supernatant to remove cells and cell debris from the cell culture medium. The isolation step involves prolonged ultracentrifugation. A potentially faster alternative is to use polyethylene glycol (PEG) instead of ultracentrifugation to isolate exosomes, a strategy used in commercially available exosome isolation kits. In this strategy, after the first centrifugation step (typically performed at 2,000 × g (RCF) for 10 minutes at room temperature), the supernatant is incubated with a polyethylene glycol (PEG) solution to precipitate vesicles. Various PEG formulations have been used. These formulations typically consist of a final PEG concentration between 5 and 15%. Additionally, salt (50–1,000 mM NaCl is preferred) may be added to the PEG solution to enhance exosome precipitation. The PEG solution is gently mixed and incubated at 4°C for 30 minutes to 16 hours. The precipitated exosomes are then pelleted by centrifugation at 10,000 × g (RCF) for 10–30 minutes at 4°C. After this step, an additional wash step using PBS, 0.9% saline, or trehalose is required to remove PEG solution residue and reduce exosome aggregation. This procedure is used in various commercially available kits, such as the Total Exosome Isolation Kit (for culture supernatants) (Thermo Fischer Scientific, USA), as an alternative to ultracentrifugation-based techniques. Therefore, this procedure does not require ultracentrifugation and, by applying low centrifugal forces, avoids the physical rupture of exosomes that is often reported in ultracentrifuge-based methods.

[0041]

[0065] The novel method for isolating exosomes includes obtaining cell culture medium from a culture of cells, removing cells and cell debris from the cell culture medium without centrifuging the cell culture medium, and subjecting the cell- and cell debris-free cell culture medium to ultracentrifugation at 3-6°C to generate a pellet containing exosomes. In some embodiments, ultracentrifugation is performed at a temperature of 4-6°C or about 4°C. The resulting pellet containing exosomes is PEG-free because PEG is not used to isolate the exosomes. Instead, exosomes are isolated by the ultracentrifugation step. Therefore, exosomes isolated according to the described method are ready for therapeutic use without the need to remove carcinogenic or other toxic byproducts, such as PEG, from commercially available exosome isolation kits. As shown in the Examples and Table 2, the disclosed method results in a much higher yield of exosomes per mL of cell culture supernatant than commercially available exosome isolation kits. Therefore, the disclosed method is more suitable for isolating large numbers of exosomes for therapeutic purposes compared to methods known in the prior art.

[0042]

[0066] In certain embodiments, a method for isolating exosomes from a culture of cells consists essentially of obtaining cell culture medium from the cell culture, filtering the cell culture medium to obtain a filtrate, thereby removing cells and cell debris from the cell culture medium, subjecting the filtrate to ultracentrifugation at 3-6°C to produce a pellet comprising exosomes, and optionally resuspending the pellet.

[0043]

[0067] In some embodiments, the step of removing cells and cell debris comprises filtering the cell culture medium to obtain a filtrate, which is subjected to ultracentrifugation. In certain implementations, the step of removing cells and cell debris from the cell culture medium comprises filtering the cell culture medium to obtain a filtrate, which is subjected to ultracentrifugation. In some embodiments, the cell culture medium is filtered using a 0.22 μm cellulose acetate membrane.

[0044]

[0068] In certain implementations, the filtrate is subjected to ultracentrifugation at a speed of 99,650 to 100,350 xg (RCF) for at least 40 minutes, such as between 40 and 70 minutes or about 60 minutes. In some embodiments, ultracentrifugation is at a speed of 99,850-100,230×g (RCF), 99,850-100,150×g (RCF), 99,880-100,120×g (RCF), 99,900-100,100×g (RCF), 99,988-100,195×g (RCF), 99,976-100,200×g (RCF), 99,967-100,206×g (RCF), 99,916-100,255×g (RCF), 99,944-100,237×g (RCF), or 99,913-100,262×g (RCF). In certain implementations, the filtrate is subjected to ultracentrifugation at a speed of 100,000 to 100,100 × g (RCF) or 100,000 × g (RCF). In such implementations, the filtrate is subjected to ultracentrifugation for 60 minutes, preferably at 4° C. In certain embodiments, ultracentrifugation is performed in a centrifuge equipped with a swing rotor that can hold centrifuge tubes with a capacity of at least 15 mL.

[0045]

[0069] In some embodiments, the described methods isolate exosomes from hIDPSCs. Such methods include culturing hIDPSCs in a basal medium, replacing the basal medium with serum-free medium when the hIDPSCs reach at least 80% confluence, and recovering the serum-free medium after the hIDPSCs have been cultured in the serum-free medium for at least 12 hours. In some implementations, the serum-free medium is recovered after the hIDPSCs have been cultured in the serum-free medium for 48 hours or less. In some embodiments, the basal medium is replaced with serum-free medium when the hIDPSCs are not more than 90% confluent. The methods further include removing cells and cell debris from the recovered serum-free medium, including filtering the recovered serum-free medium to obtain a filtrate, but not centrifuging the recovered serum-free medium, ultracentrifuging the filtrate at 3-6°C to generate a pellet containing exosomes, and optionally resuspending the pellet. The pellet may be resuspended in saline, for example, sterile 0.9% saline and / or isotonic saline.

[0046]

[0070] In a specific implementation of isolating exosomes from hIDPSCs, the method involves: i) culturing cells at 80-90% confluence, and then rehydrating the cells with the basal medium of these cells, which is Dulbecco's Modified Eagle Medium (DMEM) / Ham's F12 (DMEM / F12) supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, and 2 mM non-essential amino acids (all from Gibco, Carlsbad, USA); ii) replacing the serum-free medium with Dulbecco's Modified Eagle Medium (DMEM) / Ham's F12 (DMEM / F12) supplemented with 100 units / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, and 2 mM non-essential amino acids (all obtained from Gibco, Carlsbad, USA); iii) filtering the collected serum-free medium (Dulbecco's Modified Eagle Medium (DMEM) / Ham's F12 (DMEM / F12) supplemented with 100 units / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, and 2 mM non-essential amino acids, all obtained from Gibco, Carlsbad, USA) through a Corning® 250 mL vacuum filter (Corning, New York, USA) fitted with a 0.22 μm cellulose acetate membrane to remove cellular debris from the sample. iii) subjecting the filtrate to a single ultracentrifugation step at 100,000 x g (RCF) for 60 minutes at 4°C to recover the exosomes; and vi) recovering the exosome pellet.

[0047]

[0071] As shown in Table 1, the methods described herein differ from known methods by avoiding excessive centrifugation steps that can compromise exosome integrity. Compared to alternative exosome isolation methods, the methods described herein, which avoid the use of PEG, allow for the use of isolated exosomes in therapeutic applications.

[0048]

[0072] Furthermore, the method described in the present invention allows for the isolation of exosomes (such as from hIDPSCs) at a high yield when compared to other previously described methods, e.g., approximately 0.86 x 10 exosomes in the culture medium. 10 In some embodiments, it was possible to obtain at least 1 x 10 exosomes / mL of filtered culture medium. 10 Furthermore, the disclosed method involves using a swing rotor to separate exosomes at a flow rate of 15 mL (5 mL x 3 rotor P65ST, Hitachi Koki, Japan) to 216 mL (36 mL x 6 rotor type AH-629(36) - Sorvall, USA) per hour, resulting in approximately 1.3 x 10 11 (Using 15 mL of culture medium) ~1.8 × 10 12 The method is easily scalable using a swing-wheel rotor, allowing for the production of exosomes per hour (using 210 mL of culture medium). The disclosed method has been shown to ensure the purity, integrity, and stability of exosome cargo (protein and RNA) for 90 days. The simplicity of the method, requiring a single ultracentrifugation step, combined with the high yield and purity of exosomes, are key advantages for the therapeutic use of these vesicles in cell-free therapies (reviewed by Araldi et al., 2020. Cells, Vol. 9(2663):1-29).

[0049] [Table 1]

[0050]

[0073] Exosomes isolated by the described methods have a diameter of 10 to 200 nm, e.g., 20 to 150 nm or 40 to 120 nm. In some embodiments, exosomes have a diameter of 20 to 60 nm. In some embodiments, the isolated exosomes express at least one marker selected from integrin, intercellular adhesion molecule 1 (ICAM-1), epithelial cell adhesion molecule (EpCAM), CD31, annexin, TSG01, apoptosis-related gene-interacting protein X (ALIX), Rab5b, HLA-G, HSP70, CD63, lysosomal-associated membrane protein 2 (LAMP2), and lysosomal integral membrane protein (LIMP). In certain embodiments, the at least one marker is CD63. In other embodiments, the isolated exosomes contain proteins, lipids, cytosolic components, and nucleic acids. In certain embodiments, the nucleic acids include miRNA and lncRNA.

[0051]

[0074] The exosomes obtained using the methods proposed in the present invention are formulated for therapeutic use by combining with sterile isotonic saline (0.9% saline), which does not impart any type of toxicity and therefore makes the final product pharmaceutically acceptable. The exosomes may be formulated for administration by, but not limited to, local, intranasal, intravenous, or intrathecal injection.

[0052]

[0075] In other embodiments, exosomes are formulated for administration by routes including, but not limited to, intranasal, intravenous, and intrathecal. 0.9% sterile saline was used as the carrier for all routes. These routes ensure efficient delivery of exosomes to the central nervous system (CNS), making them ideal for treating neurodegenerative diseases.

[0053]

[0076] Suitable methods for administering therapeutic compositions according to the methods of the presently disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravenous, intramuscular, and / or intra-arterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, intratracheal introduction, surgical implantation, transdermal delivery, local injection, intranasal delivery, and ultrafast injection / bombardment. Where applicable, continuous infusion can enhance drug accumulation at the target site (see, e.g., U.S. Patent No. 6,180,082).

[0054]

[0077] The components of the present invention may also be administered parenterally, i.e., subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly, or intraperitoneally, as an injectable dose of exosomes, preferably in a physiologically acceptable diluent, with or without a pharmaceutical carrier, which may be a sterile liquid such as water, saline, aqueous dextrose and related sugars, alcohols such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters, or fatty acid glycerides, or acetylated fatty acid glycerides, or a mixture of such liquids, with or without a pharmaceutically acceptable surfactant such as a soap or detergent, a suspending agent such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or an emulsifying agent and other pharmaceutical adjuvants.

[0055]

[0078] The exosomes according to the present invention can be incorporated into the aforementioned dosage forms. This can be achieved by mixing with a pharmaceutically suitable excipient. Pharmaceutically suitable excipients include, inter alia, fillers and carriers (e.g., cellulose, microcrystalline cellulose (e.g., Avicel®, etc.), lactose, mannitol, starch, calcium phosphate (e.g., Di-Cafos®, etc.)), ointment bases (e.g., yellow petrolatum, paraffin, triglycerides, waxes, wool fat, wool fat alcohol, lanolin, hydrophilic ointments, polyethylene glycol), bases for suppositories (e.g., polyethylene glycol, cocoa butter, hydrogenated fat), solvents (e.g., water, ethanol), and the like. alcohol, isopropanol, glycerol, propylene glycol, medium chain triglyceride fatty oils, liquid polyethylene glycol, paraffin), surfactants, emulsifiers, dispersing agents or wetting agents (e.g., sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols (e.g., Lanette®, etc.), sorbitan fatty acid esters (e.g., Span®, etc.), polyoxyethylene sorbitan fatty acid esters (e.g., Tween®, etc.), polyoxyethylene fatty acid glycerides (e.g., Cremophor®, etc.), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers (e.g., Pluronic®, etc.), buffer solutions, acids and bases (e.g., phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine), isotonicity agents (e.g., glucose, sodium chloride), adsorbents (e.g., highly dispersed silica), viscosity-increasing agents (e.g., gel-forming agents, thickeners and / or binders (e.g., polyvinylpyrrolidone, methylcellulose, triallose, hydroxypropylmethylcellulose, hydroxypropyl-cellulose, sodium carboxymethylcellulose, starch, carbomer, polyacrylic acid (e.g., Carbopol®, etc.), alginates, gelatin), disintegrants (e.g., modified starch, sodium carboxymethylcellulose, sodium starch glycolate (e.g., Explotab®, etc.),Crosslinked polyvinylpyrrolidone, croscarmellose sodium (e.g., AcDiSol®, etc.), flow regulators, lubricants, glidants and release agents (e.g., magnesium stearate, stearic acid, talc, highly dispersed silica (e.g., Aerosil®, etc.)), coating materials (e.g., sugar, shellac) and film formers for fast-dissolving or controlled-dissolving films or diffusion membranes (e.g., polyvinylpyrrolidone (e.g., Kollidon®, etc.), polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, hydroxypropyl methylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates (e.g., Eudragit®, etc.)), capsule materials (e.g., gelatin, hydroxypropyl methylcellulose), synthetic polymers (e.g., polylactic acid, polyglycolide, polyacrylate, polymeth ... Examples of suitable anti-inflammatory agents include acrylates, polymethacrylates (e.g., Eudragit®, etc.), polyvinylpyrrolidone (e.g., Kollidon®, etc.), polyvinyl alcohol, polyvinyl acetate, polyethylene oxide, polyethylene glycol, and copolymers and block copolymers thereof), plasticizers (e.g., polyethylene glycol, propylene glycol, glycerol, triacetin, triacetyl citrate, dibutyl phthalate), penetration enhancers, stabilizers (e.g., antioxidants such as ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, etc.), preservatives (e.g., parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate), colorants (e.g., inorganic pigments such as iron oxide, titanium dioxide, etc.), hyaluronic acid, fragrances, sweeteners, flavor- and / or odor-masking agents.

[0056]

[0079] For clinical use, exosome solutions (in 0.9% sterile saline) can be stored at -20°C to -80°C for up to 90 days without affecting exosome integrity or cargo. Furthermore, unlike cell products whose storage protocols require the use of cryogenic stabilizers such as dimethyl sulfoxide (DMSO), glycerol, trehalose, or polyhydroxylated alcohols, exosome storage does not require the addition of any cryogenic stabilizers, enhancing the biosafety of our product.

[0057]

[0080] RNA sequencing (RNA-Sep) leverages the capabilities of high-throughput sequencing methods to gain insights into the cellular transcriptome. RNA-Seq offers high coverage and superior resolution of the dynamic nature of the transcriptome. RNA-Sep is generally reliable for accurately measuring changes in gene expression levels. Therefore, it is a suitable method for designing therapeutic compositions composed of exosomes based on comparative transcriptome analysis. Based on Figure 15B, analyzing two different samples from NestaCell and NestaExo products, we can conclude that NestaExo must share a minimum of 2,000 genes with a maximum of 4,000 that are upregulated by both NestaCell and NestaExo. Similarly, NestaExo must share a minimum of 20,000 genes that are downregulated by both NestaCell and NestaExo.

[0058]

[0081] Comparative transcriptome analysis revealed that NestaCell and NestaExo share transcriptional profiles with human embryonic stem cells (hESCs) and brain cells, and thus the NestaExo composition should contain at least 55% of the genes expressed by hESCs and human brain cells, as well as 11% to 20% of dentin genes (Figure 16). Meanwhile, NestaCell and NestaExo represent between approximately 2.27% and approximately 5.20% of dental and mesenchymal stem cell (MSC) genes. At the same time, the transcriptional profiles of NestaCell and NestaExo showed less similarity to hematopoietic stem cells, neural stem cells, bone marrow stromal cells, and odontoblasts. We refined our search by the site of expression in the nervous system—brain cells (Figure 16). The NestaCell and NestaExo therapeutic compositions demonstrated high transcriptional similarity with genes whose expression was observed in the hippocampus (approximately 47%-59%), cerebellum (approximately 63%-59%), cerebral cortex (approximately 45%-55%), and cerebrospinal fluid (approximately 31%-55%). Brain regions such as the amygdala, substantia nigra, caudate nucleus, thalamus, and hypothalamus showed transcriptional profile similarities of approximately 0.2% to 6% with NestaCell and NestaExo (Figure 16).

[0059]

[0082] Transcription factors (TFs) are regulatory molecules-proteins that control the rate of transcription of genetic information from DNA to messenger RNA. The function of TFs is to turn genes on and off, ensuring that those genes are expressed in the right amounts at the right time in the desired cells throughout the life of the cells.

[0060]

[0083] We analyzed the expression percentage of various transcription factors we discovered in NestaCell and NestaExo. CTCF is a highly conserved zinc finger protein. It can function as a transcriptional activator, repressor, or insulator protein. Early growth response factor 1 (EGR1) is a transcription factor primarily involved in tissue injury, immune responses, and fibrotic processes. The transcription factor Sp4 regulates dendritic patterning during cerebellar maturation. Sp1 is a transcription factor that responds to inflammatory signals generated in Alzheimer's disease (AD) brains. Kruppel-like factor 7 (KLF7) is a transcription factor (TF) that promotes axonal regeneration in the central nervous system. All of these TFs showed high expression percentages in NestaCell and NestaExo (Figure 17). SP1 accounts for 43% to 55% of the transcriptome in NestaCell and NestaExo, whereas KLF7 accounts for approximately 27% to 34%, SP4 for approximately 29% to 34%, EGR1 for approximately 24% to 30%, and CTCF for approximately 16% to 20%.

[0061]

[0084] Using KEGG term enrichment analysis based on the list of commonly identified non-coding RNA lncRNA transcripts and the list of exclusively identified non-coding RNA lncRNA transcripts as differentially expressed (DE) in exosomes from two hIDPSC donors, we demonstrated that NestaExo exosomes can be used for the prevention and treatment of neurodegenerative disorders, including ALS (Figure 18). In ALS, the gene ratio was found to be higher than 0.06, with a count of approximately 90. In HD, the gene ratio was found to be between 0.05 and 0.06, with a count of 80.

[0062]

[0085] KEGG term enrichment analysis was also performed based on the microRNA transcript list of non-coding RNAs (Figure 19). These results further demonstrate that NestaExo exosomes can be used for the prevention and treatment of neurodegenerative disorders. In ALS, the gene ratio was higher than 0.11, with a count of approximately 14. In HD, the gene ratio was 0.10, with a count of approximately 12. Analysis using KEGG overrepresented pathways revealed that the gene ratio for the neurodegenerative (multiple disease) pathway was approximately 0.1, with a count of approximately 30. In coronavirus disease, the gene ratio was higher than 0.075, with a count of approximately 30. In lifespan regulation pathways, the gene ratio was between 0.025 and 0.050, with a count of approximately 10. [Example]

[0063] Example 1 Exosome isolation from the culture supernatant of human immature dental pulp stem cells (hIDPSC)

[0086] Exosomes were isolated from the culture supernatant of human immature dental pulp stem cells (hIDPSCs) obtained from two different donors and cultured according to patent number US9790468.

[0064]

[0087] hIDPSCs were cultured in complete basal culture medium until they reached 80-90% confluence. The complete medium was then replaced with serum-free medium and incubated under these conditions for 24 hours. This step is essential for recovering exosomes released into the culture medium.

[0065]

[0088] Considering that the therapeutic potential of exosomes may depend on the stem cell properties of hIDPSCs, and because exosomal content can vary depending on the state of cell differentiation, we analyzed the effects of 24 hours of serum starvation. Cells were stored in this supernatant for 24 hours. Results showed that serum starvation (24 hours) did not alter the MSC marker pattern of hIDPSCs, and hIDPSCs remained attached to plastic and exhibited a fibroblast-like morphology (Figure 1B), similar to hIDPSCs maintained in complete medium (i.e., fetal serum) (Figure 1A). Furthermore, hIDPSCs cultured under serum starvation were negative for the biomarkers CD11B (Figure 2A), CD19 (Figure 2B), CD34 (Figure 2C), CD45 (Figure 2D), CD80 (Figure 2E), CD86 (Figure 2F), and HLA-DR (Figure 2G), but positive for the biomarkers CD73 (Figure 3A), CD90 (Figure 3B), CD105 (Figure 3C), and CD146 (Figure 3D), demonstrating that serum starvation (24 h) does not alter the immune profile of hIDPSCs according to the minimal criteria for defining multipotent MSCs proposed by the International Society for Cellular Therapy.

[0066]

[0089] Once it was clear that 24 hours of serum starvation had no effect on the immune profile of hIDPSCs, the supernatant was collected by aspiration and filtered using a Corning® 250 mL vacuum filter fitted with a 0.22 μm cellulose acetate membrane. To prevent any contamination, these procedures were performed in a vertical laminar flow. To preserve the exosomal content, the filtrate was immediately stored at -80°C in a 250 mL storage bottle system connected to a Corning® 250 mL vacuum filter. At the time of isolation, the filtrate was thawed at room temperature, transferred to a 13 mL ultracentrifuge tube, and subjected to ultracentrifugation at 100,000 × g (RCF) for 60 minutes at 4°C using a CP90WX ultracentrifuge (Hitachi Koki Himac, Japan). The supernatant was aspirated and discarded. The exosome pellet was resuspended in 100 μL of 0.9% sterile saline.

[0067]

[0090] To confirm exosome isolation, 10 μL of the exosome pellet was immediately analyzed by transmission electron microscopy (TEM) using a total magnification of 120,000x with negative staining using uranyl acetate. This analysis was performed using a LEO 906E electron microscope (Zeiss, Germany). The results showed the presence of spherical nanoparticles with diameters between 20 and 60 nm, consistent with exosomes (Figure 4A).

[0068]

[0091] To confirm the isolation of exosomes and evaluate the integrity of these vesicles, 20 μL of the exosome suspension was transferred to a mica surface, dried in a vacuum chamber, and subjected to surface scanning using a MultiMode8 (Brucker, Germany) high-resolution atomic force microscopy (AFM). The results revealed the presence of a large number of spherical nanoparticles of approximately 6.2 nm, which corresponds to the expected diameter for exosomes in the dry state (Figures 5A and 7A). To confirm this result, the liquid exosome pellet was also subjected to atomic force microscopy using a JPK NanoWizard AFM (JPK-Brucker, Germany). The results showed the presence of a large number of spherical nanovesicles of approximately 100 nm (Figure 6A), confirming that the disclosed method is capable of successfully isolating exosomes.

[0069]

[0092] Evidence that the disclosed method is useful for isolating exosomes from hIDPSCs, although not limited to hIDPSCs, is provided below, where the exosome concentration and size distribution profile (polydispersity index) were measured using a NanoSight NS300 nanoparticle tracking analyzer (Malvern Paranlytical, UK). The results showed that the disclosed method can successfully isolate vesicles with a diameter of approximately 100.1 ± 1.2 nm (Figure 8C), which matches the diameter of exosomes. In addition to the disclosed method being simpler, requiring only a single step consisting of 60 minutes of ultracentrifugation to isolate exosomes from culture medium, the NTA results also showed that the disclosed method was able to isolate approximately 1.0 × 10 exosomes from each 13 mL of culture medium. 11 We also demonstrated the isolation of exosomes (Figure 8B). As shown in Table 2, the disclosed method isolates over 1,000-fold more exosomes than commercially available exosome isolation kits, enabling large-scale production of exosomes for therapeutic use.

[0070]

[0093] Furthermore, the disclosed method preserves the integrity (Figure 8A) and purity (Figures 5A, 6A, and 7A) of hIDPSC-derived exosomes. The disclosed method also ensures the isolation of exosomes free of contaminating apoptotic bodies (extracellular vesicles with diameters between 1 and 5 μm, i.e., between 1,000 and 5,000 nm). This is evidenced by repeated NTA analysis (Figure 8B) revealing that 90% of isolated exosomes (D90) had a diameter of 137.1 nm, as further evidenced by the polydispersity plot (Figure 8C). To confirm that the disclosed method guarantees the isolation of pure exosomes, isolated vesicles were subjected to immunodetection of the exosome marker CD63 using image flow cytometry. As shown in Figures 9A and 9B, immunolabeling of the exosome marker CD63 was observed on the surface of 98.9% of isolated vesicles.

[0071] Example 2 Comparison of exosome isolation techniques using commercially available kits

[0094] Although ultracentrifugation-based methods are widely used to isolate exosomes, a current comparative analysis of exosome isolation methods (Patel et al., 2019, Scientific Reports, Vol. 9, p. 5335) demonstrated that polyethylene glycol (PEG)-coprecipitation-based methods, such as those using the Total Exosome Isolation Kit (Thermo Fischer Scientific), ensure the isolation of larger exosomes with the highest protein concentration, in addition to the largest exosome size. These data suggest that PEG-based coprecipitation methods can increase exosome integrity by applying lower centrifugal forces, typically 2,000 × g (RCF), which are 50-fold lower than the RCF typically applied in ultracentrifugation-based methods, typically 100,000 × g (RCF). However, this study identified the presence of PEG as an impurity in the total protein content by mass spectrometry. This result limits the therapeutic use of exosomes isolated using PEG-based methods.

[0072]

[0095] Based on these data, the disclosed method for isolating exosomes from hIDPSCs, described in this invention, was compared with two commercially available methods: (1) a method using a PEG-based co-precipitation method with the Total Exosome Isolation Kit (Thermo Fisher Scientific, USA) and (2) a method using an immunoaffinity capture-based method with the Exosome-Human CD63 Isolation / Detection Reagent (from Cell Culture Medium) (Thermo Fisher Scientific, USA). As shown in Figures 1-3, after 24 h of serum starvation, serum-free culture medium was collected in all strategies, as this time point of serum starvation did not show any changes in the immunoprofile of hIDPSCs. This culture medium was filtered using a Corning® 250 mL vacuum filter fitted with a 0.22 μm cellulose acetate membrane. To prevent any type of contamination, these procedures were performed in a vertical laminar flow. To preserve the exosomal content, the filtrate was immediately stored at -80°C in a 250 mL storage bottle system connected with a Corning® 250 mL vacuum filter.

[0073]

[0096] For exosome isolation using the PEG-based coprecipitation method, the filtrate was thawed at room temperature and then incubated with 0.5 volumes (v / v) of Total Exosome Isolation Reagent (contained in the Total Exosome Isolation Kit for Cell Culture Media, Thermo Fisher Scientific, USA) at 4°C for 16 hours. The sample was centrifuged at 10,000 × g (RCF) for 60 minutes using a Legend X1R refrigerated centrifuge and a TX-400 rotor (Thermo Fisher Scientific, USA). The supernatant was then aspirated and discarded, and the exosome pellet was resuspended in 100 μL of 0.9% sterile saline and stored at -20°C.

[0074]

[0097] For exosome isolation using an immunoaffinity capture method, exosome suspensions previously obtained using the Total Exosome Isolation Kit were homogenized and incubated with Dynabeads contained in the Exosome-Human CD63 Isolation / Detection Reagent (Thermo Fisher Scientific, USA) at 4°C for 16 hours. After incubation, exosomes (expressing the canonical marker CD63) were isolated using a DynaMag-2 magnetic separator (Thermo Fisher Scientific, USA). The exosomes were then resuspended in 100 μL of 0.9% sterile saline.

[0075]

[0098] To confirm the presence and integrity of exosomes isolated using both commercially available methods, the samples were negatively stained with uranyl acetate and subjected to transmission electron microscopy as described in Example 1. The results demonstrated that both the PEG-based co-precipitation method and the immunoaffinity capture-based method were capable of isolating exosomes, as shown in Figures 4B and 4C, respectively. However, exosomes isolated using the PEG-based method (Figure 4B) and the immunoaffinity capture-based method (Figure 4C) were aggregated into clumps of PEG, whereas the disclosed method does not require the use of these chemicals to co-precipitate vesicles (Figure 4A), and therefore exosomes isolated using the disclosed method are completely free of co-precipitating polymer residues.

[0076]

[0099] To assess exosome integrity and analyze the presence of co-precipitating residues, exosomes isolated using commercially available methods were subjected to atomic force microscopy (AFM) analysis. Dry-state AFM analysis confirmed the presence or absence of PEG contamination in exosome samples isolated using the PEG-based co-precipitation method (Figure 5B) and the immunoaffinity capture-based method (Figure 5C). These results were also confirmed by liquid-state AFM, as shown in Figures 6B and 6C. 3D AFM analysis also confirmed PEG precipitation (Figure 7B). In contrast, the presence of any precipitates was not detected in exosomes isolated using the disclosed method (Figures 5A, 6A, and 7A). These data suggest that by not using any precipitating chemicals and using serum-free medium, the disclosed method ensures the purity and integrity of hIDPSC-derived exosomes.

[0077] [Table 2]

[0078] Example 3 hIDPSC-derived exosomes (NestaExo) can be stored for up to 90 days without affecting their protein and RNA cargo

[0100] Considering the potential clinical use of hIDPSC-derived exosomes as therapeutic agents for, but not limited to, neurodegenerative disorders and COVID-19, we analyzed the stability of these extracellular vesicles.

[0079]

[0101] To this end, hIDPSC-derived exosomes isolated using the disclosed method were stored in 0.9% saline at three different thermal conditions (+4°C, -20°C, and -80°C) for 90 days. The exosomes were then thawed at room temperature and subjected to nanoparticle tracking analysis. The results showed that 90 days of storage at +4°C, -20°C, and -80°C did not affect exosome morphology, maintaining their medium diameters of 93.1 nm (+4°C, Figures 10A and 10B), 98.0 nm (-20°C, Figures 10C and 10D), and 94.3 nm (-80°C, Figures 10E and 10F), suggesting that storage of hIDPSC-derived exosomes at these temperatures does not alter the morphological characteristics of these extracellular vesicles. However, NTA results showed that exosomes derived from hIDPSCs stored at +4°C for 90 days were significantly higher than those stored at -20°C (1.28 × 10 11 ±2.46×10 9 exosomes / mL, Figure 10C) and −80 °C (1.43 × 10 11 ±1.02×10 10 The exosome concentration (7.16 × 10 exosomes / mL, Figure 10E) was significantly higher than the final exosome concentration observed in the samples stored at 100 μg / mL. 9 ±1.22×10 8 These data were confirmed by analyzing exosomes obtained from two different donors.

[0080]

[0102] Considering the NTA results for storage of hIDPSC-derived exosomes at -20°C and the benefit of this temperature for facilitating the transport of exosomes for clinical purposes without the need for an ultrafreezer, freshly isolated exosomes and exosomes stored at -20°C for 90 days from hIDPSCs of two donors were subjected to protein isolation using Pierce™ RIPA buffer (Thermo Fisher Scientific, USA). Total protein concentration was measured using the bicinchoninic acid method (BCA) with a BCA Pierce™ kit (Thermo Fisher Scientific, USA). After measuring the total protein concentration in both samples of freshly isolated exosomes (981.2 and 1,152.0 μg protein / mL, respectively) and exosomes stored at -20°C for 90 days (981.4 and 1,168.4 μg protein / mL, respectively), 50 μg of exosomal protein was subjected to electrophoretic analysis on an SDS-PAGE gel. The results showed very similar electrophoretic profilers between the freshly isolated exosomes and exosomes stored at -20°C for 90 days (Figure 11), demonstrating that long-term storage does not induce changes in protein content.

[0081]

[0103] Considering that exosomes also transport coding RNA (messenger RNA - mRNA) and non-coding RNA (long non-coding RNA - lncRNA and small RNA) that may also confer therapeutic activity, freshly isolated exosomes and exosomes stored at -20°C for 90 days were subjected to RNA extraction using TRIZol reagent (Thermo Fisher Scientific, USA). The isolated total RNA was subjected to quality control (QC) using the BioAnalyzer platform (Agillent, USA). The results show that samples from both freshly isolated exosomes (1,144 ng / μL, Figure 12A) and exosomes stored at -20°C for 90 days (1,444 ng / μL, Figure 12B) maintained similar RNA concentrations and exhibited RNA integrity numbers (RINs) higher than 6 (8.5 for freshly isolated exosomes, Figure 12A, and RIN 6.2 for exosomes stored at -20°C for 90 days, Figure 12B), suggesting that storage at -20°C for 90 days does not affect RNA concentration or quality.

[0082] Example 4 Scaling up exosome production

[0104] Exosomes were successfully isolated from input samples in increasingly larger sizes, e.g., 6-well dishes, 60- to 100-cm well plates, and flasks ranging from T-150, T-175, and T-225 flasks (Corning). The exosomes isolated in each case showed similar quality and integrity, e.g., a mean size of approximately 95 nm.

[0083]

[0105] To demonstrate that the disclosed method is scalable, conditioned culture medium from hIDPSCs was subjected to exosome isolation using the method described by the inventors using an Optima XE-900 ultracentrifuge (Beckman Coulter Inc., UK) equipped with an SW28Ti Swinging-Bucket Aluminum rotor for six 38.5 mL ultracentrifuge tubes (allowing for ultracentrifugation of 231 mL of filtered conditioned culture medium / hour).

[0084]

[0106] Exosomes (NestaExo) isolated using this ultracentrifuge (with a total volume capacity of 231 mL) were compared with NestaExo isolated using a CP100WX ultracentrifuge equipped with a swing-out rotor for a total volume capacity of 78 mL (6 × 13 mL). The exosome pellet was resuspended in 500 μL (pellets obtained using the Optima XE-900 ultracentrifuge) or 200 μL (pellets obtained using the CP100WX ultracentrifuge) of saline.

[0085]

[0107] Both exosome pellets were subjected to total protein extraction using RIPA lysis and extraction buffer (Thermo Fisher Scientific, Carlsbad, CA, USA, Cat. no. 89900) according to the manufacturer's instructions. Relative protein concentrations were measured by the bicinchoninic acid (BCA) method using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's instructions. The assay was performed in sextuplicate (due to the rotor's capacity for six tubes). We then calculated the average protein concentration and normalized this value by the volume of saline in which each exosome pellet was resuspended.

[0086]

[0108] The exosome solution isolated using the OptimaXE-90 centrifuge had a normalized average concentration of 2,537.33 μg / mL, while the exosome solution isolated using the CP100WX centrifuge had an average concentration of 2,644.10 μg / mL. These results demonstrate that the disclosed method can consistently obtain exosomes (relative protein mass, μg) per milliliter of conditioned culture medium (mL).

[0087]

[0109] In addition, the method is easily scalable. Considering that isolation depends on the capacity of the ultracentrifuge bottle and the use of a rotor capable of reaching 100,000 × g (RCF), the method yields approximately 1.3 × 10 11 (Use 15 mL of culture medium; 5 mL × 3 - Rotor P65ST, Hitachi Koki, Japan) ~ 1.8 × 10 12 Exosomes could be isolated per hour (using 210 mL of culture medium; 36 mL x 6 rotor type AH-629(36) - Sorvall, USA), enabling the use of this method as a procedure to obtain exosomes with purity and integrity for clinical purposes.

[0088] Example 5 NestaExo demonstrates therapeutic potential against multiple neurodegenerative disorders, COVID-19, and cancer

[0110] Considering that the therapeutic potential of exosomes is mediated by the transcripts delivered to recipient cells by these extracellular vesicles, we performed unbiased transcriptome analysis using high-throughput sequencing (RNA-Sep). To this end, total RNA from hIDPSCs obtained from two different donors and hIDPSC-derived exosomes were isolated using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA quality and concentration were analyzed using a BioAnalyzer (Agillent, USA). RNA-Seq libraries were prepared using the TrueSeq Stranded mRNA Sample Prep Kit (Illumina, USA) and sequenced on an Illumina HiSeq200 (Illumina, USA), generating approximately 20 million reads for each sample. Raw data were downloaded as FASTQ files. The RNA-Seq libraries were subjected to quality control (QC) and analysis using a custom pipeline. All sequencing libraries were quality trimmed and adapter trimmed using trimmomatic, then assessed for high quality using fastqc and MultiQC. Trimmed reads were mapped to the human genome (hg19) and transcriptome (Esembl, release 72) using the software STAR. Gene expression levels were calculated using the FPKM metric (fragments per kilobase of exon per million fragments mapped) using Cuffdiff (v. 2.2.0). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEEG) term enrichment analysis was performed based on a normalized list of mRNAs and small RNAs commonly identified in hIDPSC-derived exosomes.

[0089] The FASTQC report revealed that the raw data obtained from RNA-Seq for all samples showed high-quality scores after trimming of Illumina adapter sequences, as demonstrated in Figure 13. After alignment using STAR software, QualiMap results showed that 70% of the reads from the hIDPSC samples (Figure 14A) and 56% of the reads from the hIDPSC-derived exosomes (NestaExo) (Figure 14B) were located within exon regions. A quantitative Venn diagram showed that the transcriptomes of hIDPSCs (NestaCell) and hIDPSC-derived exosomes (NestaExo) were highly similar, with the cells (hIDPSCs) and exosomes expressing 5,930 transcripts in common (Figure 15). Comparative transcriptome analysis showed that hIDPSCs (NestaCell) and hIDPSC-derived exosomes (NestaExo) possess unique transcriptome profiles with varying percentages of similarity to the transcriptomes of various regions of the central nervous system, such as the spinal cord, cerebral cortex, and hippocampus (Figure 16). Further supporting this finding, over 50% of the transcripts identified in hIDPSCs (NestaCell) and hIDPSC-derived exosomes (NestaExo) are present in regions affected by neurodegenerative diseases: the hippocampus and cerebellum for NestaCell, and the cerebral cortex along with the hippocampus and cerebellum for NestaExo (Figure 16). Further evidence of NestaExo specificity is that the transcriptome of hIDPSC-derived exosomes (NestaExo) shares over 55% of the transcripts expressed by embryonic stem cells (ESCs), which possess limited differentiation potential (pluripotency).

[0090]

[0111] Furthermore, RNA-Seq analysis also demonstrated that hIDPSC-derived exosomes possessed a unique molecular profile, containing 74 genes out of 375 uniquely expressed by hIDPSCs (NestaCell products), conferring neuroprotective and neuroregenerative properties to NestaExo (Table 3), providing ample evidence for transcriptome similarity with brain regions (Figure 16). Given the high similarity of the transcriptome of hIDPSC-derived exosomes (NestaExo) to brain (Figure 16), we identified the mRNAs of transcription factors present at high levels in these vesicles (NestaExo). Results showed that two batches of NestaExo contained high levels of the nervous system transcription factors NFIC, EGR1, SP1, SP4, and Klf7 (Figure 17). Interestingly, these same factors were also found to be overexpressed in hIDPSCs (NestaCell) (Figure 17), suggesting that exosomes naturally released by these cells are associated with the clinical improvement already demonstrated in Huntington's disease patients treated with hIDPSCs (NestaCell) in a phase I clinical trial (Macedo et al., 2021. Cytotherapy, Vol. 23, 4:1).

[0091]

[0112] Based on the mRNAs present at the highest levels in both batches of hIDPSC-derived exosomes (NestaExo), we performed KEGG term enrichment analysis. The results showed that the mRNAs delivered by NestaExo could regulate pathways related to amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and adherens junctions that are disrupted during metastatic processes (Figure 18), demonstrating the application of NestaExo as a therapeutic agent for neurodegenerative disorders and cancer.

[0092]

[0113] Considering that the therapeutic potential of MSC-derived exosomes may also be mediated by lncRNAs (Born et al., 2020, Bioengineering & Translational Medicine, Vol. 5, No. 3, p. e10172), we identified genes targeted by lncRNAs specifically expressed in NestaExo. These genes were then subjected to enrichment analysis in KEGG terms. The results, as shown in Figure 19, indicated that genes targeted by lncRNAs specifically expressed in NestaExo are related to signaling pathways deregulated in cancer, including focal adhesions and extracellular matrix (ECM)-receptor interactions (deregulation of which is closely related to cancer metastasis) and proteoglycans (related to immune evasion) in cancer, in addition to signaling pathways deregulated in amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). These results highlight the potential of NestaExo for the treatment of neurodegenerative disorders and cancer, including, but not limited to, ALS and HD.

[0093]

[0114] Based on evidence supporting the therapeutic benefit of MSC-derived exosomes being mediated by small RNAs, particularly miRNAs (Chen et al., 2018, Journal of Immunology, Vol. 201, 8:2472–2482), we identified miRNAs specifically expressed in NestaExo, as well as genes targeted by these miRNAs. The target genes were subjected to KEGG term enrichment analysis. The results, as shown in Figure 20, revealed that miRNAs delivered by NestaExo can regulate multiple pathways found to be deregulated in cancer, including the hypoxia-inducible factor 1 (HIF-1) signaling pathway, which acts as a driver of metabolic reprogramming and is well-founded for its enrichment in pathways related to glycolysis and central carbon metabolism in cancer. These pathways are also relevant to various neurodegenerative disorders and COVID-19, including chemokine and cytokine (IL-17) signaling and interactions between cytokines and cytokine receptors and viral proteins. These results highlight that NestaExo could be explored for the treatment of neurodegenerative disorders, COVID-19, and cancer.

[0094] [Table 3-1]

[0095] [Table 3-2]

[0096] Example 6 NestaExo demonstrates therapeutic potential for the treatment of neuroblastoma Given that hIDPSCs originate from the neural crest and that exosomes derived from these cells share a transcriptomic signature with hIDPSCs that reveals therapeutic potential for the treatment of various malignancies by regulating biological processes associated with oncogenesis, such as the hypoxia-inducible factor 1 alpha (HIF-1α) signaling pathway, glycolysis, and central carbon metabolism in cancer (Figure 20), we evaluated the therapeutic potential of hIDPSC-derived exosomes (NestaExo) as a treatment for neuroblastoma (NB), a neural crest malignancy of the peripheral nervous system (Johnsen et al., 2019. Front. Mol. Neurosci. Vol. 12:1-11).

[0097] Although NB is a relatively rare disease, accounting for 6–10% of all childhood tumors, the disease accounts for 12–15% of all childhood cancer-related deaths (Johnsen et al., 2019. Front. Mol. Neurosci. Vol. 12:1–11). NB treatment diverges widely among various risk groups. Treatment for patients classified as intermediate-risk involves chemotherapy followed by surgical resection of any remaining tumor mass. Treatment for high-risk patients involves an intensive induction chemotherapy regimen followed by surgery and myeloablative therapy combined with hematopoietic stem cell reinfusion and local radiation therapy (Johnsen et al., 2019. Front. Mol. Neurosci. Vol. 12:1–11). In general, patients in the intermediate-risk group have a poor response to initial treatment, resulting in disease recurrence. In high-risk patients, persistence of neuroblastoma cells in the bone marrow after induction chemotherapy is also associated with poor prognosis (Johnsen et al., 2019. Front. Mol. Neurosci. Vol. 12:1-11). Therefore, novel therapeutic approaches that can slow the progression of NB in ​​both intermediate- and high-risk patients may improve its prognosis.

[0098] Based on this, neuroblastoma-derived cell line SH-SY5Y (ATCC CRL-2266) was incubated at various concentrations, relative weight, at 50 μg (5×10 9 equivalent to 6.25 (6.255 × 10 exosomes / mL) 8The cells were treated with hIDPSC-derived exosomes (NestaExo) at a dose of 50 μg (equivalent to 5 × 10 exosomes / mL) for 48 hours. Cell viability was then analyzed using Cell Count Kit 8 (CCK8, Sigma-Aldrich, Saint Louis, USA). Results were compared with the maximum dose of 50 μg (5 × 10 exosomes / mL). 9 The results showed that NestaExo-treated cells (equivalent to exosomes / mL) were able to reduce cell proliferation compared to the control (cells not treated with NestaExo) (Figure 21).

[0099] In another strategy, SH-SY5Y cell lines were treated with two doses of NestaExo (50 and 6.25 μg of hIDPSC-derived exosomes) for 48 hours. Cells were lysed using Pierce™ RIPA buffer (Thermo Fischer Scientific, Carlsbad, USA), and the protein lysates were subjected to protein concentration analysis using the Pierce™ BCA Protein Assay (Thermo Fischer Scientific, Carlsbad, USA). The expression of 84 cancer-related proteins was simultaneously analyzed in cell lysates (200 μg of total protein) of SH-SY5Y cells treated with 50 and 6.25 μg of NestaExo using the Proteome Profiler™ Array-Human XL Oncology Array Kit (R&D Systems, Miami, USA, reference number ARY026). The results showed that NestaExo was able to down-regulate the expression levels of 12 cancer-related proteins, 9 of which were dose-dependent (Figure 22). Among the down-regulated proteins was endoglin, also known as CD105 (Figure 22). Endoglin / CD105 is a transmembrane co-receptor for transforming growth factor beta (TGF-β) and bone morphogenetic protein-9 (BMP-9), and serves as a target for eliminating tumor-associated cells and enhancing the therapeutic activity of immunotherapy in neuroblastoma (Wu et al., 2019. Clin. Cancer Res., Vol. 25(15):4761-4774).

[0100] Example 7 NestaExo demonstrates therapeutic potential for Parkinson's disease

[0102] Given that RNA-seq analysis showed that NestaExo can deliver key transcripts originally identified to be deregulated in various neurodegenerative disorders, including Parkinson's disease (PD), the inventors investigated the therapeutic activity of these exosomes using preclinical models of PD.

[0101] PD is the most common neurodegenerative disorder. Epidemiological studies have shown that the prevalence of PD has doubled over the past 25 years, with a 2019 global estimate indicating that more than 8.5 million people suffer from PD (Ou et al., 2021, Front. Public health. 9:776847). Disability and death from PD are increasing more rapidly than any other neurological disorder. In 2019, the World Health Organization (WHO) estimated that PD accounted for 5.8 million disability-adjusted life years, an 81% increase since 2000, and 329,000 deaths (an increase of more than 100% since 2000) (WHO, available at https: / / www.who.int / news / item / 14-06-2022-launch-of-who-s-parkinson-disease-technical-brief).

[0102]

[0104] PD, first described by James Parkinson in 1817, is a multifactorial neurodegenerative disorder characterized by the progressive death of dopaminergic neurons in the substantia nigra (striatum) (Tofaris 2022. Cell. Mol. Life Scie. 79:2010). The dopamine deficiency caused by the loss of these neurons leads to motor dysfunction, cognitive dysfunction, and neuropsychiatric dysfunction, including anxiety and depression (Tofaris 2022. Cell. Mol. Life Scie. 79:2010).

[0103] Only 5% of PD cases are hereditary (Arena et al., 2022. Current Neurol Neurosci Reports Vol. 22:427-440). For this reason, most genetic models for PD are unrepresentative. In this regard, the neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to induce PD in rat models. This is because the 6-OHDA-induced model does not contain all PD symptoms, but it reproduces key cellular processes involved in the physiopathology of the disease, such as oxidative stress, neurodegeneration, neuroinflammation, and apoptotic neuronal death (Hernandez-Baltazar et al., 2017. Neurologia Vol. 32(8):533-539).

[0104] To evaluate the therapeutic activity of NestaExo, a total of 50 male Wistar rats weighing 230-250 g, all obtained from the Central Bioterium of Butantan Institute, were acclimated for 5 days at the Bioterium of Pharmacology Laboratory of Butantan Institute. All procedures used were approved by the Ethics Committee in Animal Use of Butantan Institute (Procedure No. 8374160321). All animals underwent stereotaxic surgery. For this, rats were anesthetized with 5% isoflurane and maintained at 3% isoflurane. The needle was inserted at the following coordinates: -1.9 mm (anteroposterior), +1.5 mm (later-lateral), and -7.7 mm (dorsoventral) from the bregma. These coordinates allowed the needle to be inserted directly into the striatum of the substancia nigra. A total of 10 animals did not receive intrastriatal injection of the neurotoxin 6-OHDA. These animals included a sham group used as a negative control. A total of 40 rats were subjected to intrastriatal injection of 6-OHDA. Ten days after surgery, all animals were subjected to an apomorphine challenge to confirm neuronal lesions. The results revealed that rats receiving intrastriatal injection of 6-OHDA increased the number of contralateral rotations (as expected) compared with the sham group (Figure 23), demonstrating that the neurotoxin promoted neuronal damage.

[0105] Ten days after the apomorphine challenge, the 6-OHDA-treated rats were assigned to four groups of 10 animals each: two groups were treated with saline (exosome vehicle) via the intravenous (IV) or intranasal (IN) route, and two groups were treated with 50 μg of NestaExo via the IV or IN route. For IV injections, a total volume of 100 μL of saline or exosome suspension (50 μg of NestaExo in 100 μL of saline) was used, whereas for IN administration, a total volume of 40 μL (20 μL per nostril) of saline or exosome suspension was used. Both IV and IN administrations were performed under anesthesia to reduce any discomfort to the animals. After 3 days (D3), 15 days (D15), and 30 days (D30) of treatment with saline (placebo) or NestaExo, the animals were subjected to locomotor and behavioral testing in an open field.

[0106] First, we analyzed the distance traveled (in centimeters) in the open field arena. As expected, the distance traveled by rats receiving intrastriatal injections of 6-OHDA and treated with saline via two routes (placebo-IV or IN) was reduced compared to the sham group. However, treatment with NestaExo via either route (IV and IN) was able to restore motor function just 72 hours or 3 days (D3) after exosome administration (Figure 24). The motor effects of NestaExo persisted for 30 days (D30) in the group of animals treated via the IV route, whereas the effects persisted for 15 days (D15) in the group treated via the IN route (Figure 24).

[0107] In another strategy, we analyzed the time rats spent in the center of the arena. This parameter provides evidence of the anxiolytic activity of exosomes. This analysis is important given that anxiety, while sometimes overlooked, is a commonly observed cognitive symptom in PD patients (Kathri et al., 2020, BIOPHA, Vol. 131:110776). Results showed that rats receiving intrastriatal injections of 6-OHDA and treated with saline (placebo—IV or IN) spent less time in the center of the arena, suggesting, as expected, higher levels of anxiety. However, treatment with NestaExo increased the time rats spent in the center of the arena to values ​​statistically similar to those of the sham group 3 days (D3) after intravenous or intranasal exosome administration (Figure 25). Interestingly, the anxiolytic activity of NestaExo was observed up to 15 days (D15) after exosome administration (via both routes—IV and IN). After 30 days of analysis (D30), we could not find any statistical difference between the groups due to increased levels of anxiety in the Sham group (Figure 25).

[0108] In another strategy, grooming time during the open field test was analyzed as an additional parameter to evaluate the anxiolytic effects of NestaExo. The results showed that rats receiving intrastriatal injections of 6-OHDA and treated with saline (placebo-IV or IN) showed reduced grooming time compared with the sham group (Figure 26), demonstrating that neurotoxicity-induced neuronal damage could mimic the anxiety observed in PD patients. However, treatment with NestaExo via the IV or IN route restored grooming time to statistically similar levels to the sham group just 3 days (D3) after exosome administration (Figure 26). The anxiolytic effects of NestaExo were observed for 15 days (D15) in both routes and for 30 days (D30) in animals treated via the IN route (Figure 26).

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Claims

1. obtaining cell culture medium from a culture of cells; removing cells and cell debris from the cell culture medium, the step comprising filtering the cell culture medium to obtain a filtrate, the step not comprising centrifuging the cell culture medium; subjecting the filtrate to ultracentrifugation at 3-6°C to produce a pellet containing exosomes, wherein the pellet does not contain polyethylene glycol; and Optionally, resuspending the pellet; 1. A method for isolating exosomes from a culture of cells, comprising:

2. Culturing the cells in serum-free medium for at least 12 hours; and harvesting the serum-free medium after the cells have been cultured in the serum-free medium for at least 12 hours, thereby obtaining cell culture medium from the cell culture. The method of claim 1 further comprising:

3. 3. The method of claim 2, wherein the cells are cultured in serum-free medium for 48 hours or less, and then the serum-free medium is harvested.

4. 10. The method of claim 1, wherein removing cells and cell debris from the cell culture medium comprises filtering the cell culture medium to obtain a filtrate.

5. 5. The method of claim 4, wherein the cell culture medium is filtered using a 0.22 μm cellulose acetate membrane.

6. 6. The method of any one of claims 1 to 5, wherein the filtrate is subjected to ultracentrifugation at a speed of 99,850 to 100,210 x g (RCF) or 99,650 to 100,350 x g (RCF) for at least 40 minutes.

7. 7. The method of claim 6, wherein the filtrate is subjected to ultracentrifugation for 40 to 70 minutes.

8. 8. The method of claim 7, wherein the filtrate is subjected to ultracentrifugation at a speed of 100,000 x g (RCF).

9. 9. The method of claim 8, wherein the filtrate is subjected to ultracentrifugation for 60 minutes.

10. The method of claim 10, wherein the exosomes in the pellet have a diameter of about 40 to 120 nm.

11. obtaining cell culture medium from the cell culture; filtering the cell culture medium to obtain a filtrate, thereby removing cells and cell debris from the cell culture medium; subjecting the filtrate to ultracentrifugation at 3-6°C to generate a pellet containing exosomes; and Optionally, resuspending the pellet; 8. The method of claim 7, consisting essentially of:

12. The method of claim 11, wherein the exosomes in the pellet have a diameter of about 20 to 150 nm.

13. 1. A method for isolating exosomes from human immature dental pulp stem cells (hIDPSCs), comprising: Culturing hIDPSCs in a basal medium; replacing the basal medium with serum-free medium when the hIDPSCs reach at least 80% confluence; harvesting the serum-free medium after the hIDPSCs have been cultured in the serum-free medium for at least 12 hours; removing cells and cell debris from the recovered serum-free medium, the step comprising filtering the recovered serum-free medium to obtain a filtrate, the step not comprising centrifuging the recovered serum-free medium; subjecting the filtrate to ultracentrifugation at 3-6°C to generate a pellet containing exosomes; and Optionally, resuspending the pellet; The above method, comprising:

14. 14. The method of claim 13, wherein the basal medium is replaced with serum-free medium when the hIDPSCs are not more than 90% confluent.

15. 14. The method of claim 13, wherein the hIDPSCs are cultured in serum-free medium for no more than 48 hours, after which the serum-free medium is harvested.

16. 14. The method of claim 13, wherein removing cells and cell debris from the harvested serum-free medium comprises filtering the harvested serum-free medium to obtain a filtrate.

17. 15. The method of claim 14, wherein the cell culture medium is filtered using a 0.22 μm cellulose acetate membrane.

18. 18. The method of any one of claims 13 to 17, wherein the serum-free medium is Dulbecco's Modified Eagle's Medium (DMEM) / Ham's F12 (DMEM / F12) containing 100 units / mL penicillin, 100 μg / mL streptomycin, 2 nM L-glutamine, and 2 mM non-essential amino acids.

19. 18. The method of any one of claims 13 to 17, wherein the filtrate is subjected to ultracentrifugation at a speed of 99,850 to 100,210 x g (RCF) or 99,650 to 100,350 x g (RCF) for at least 40 minutes.

20. 20. The method of claim 19, wherein the filtrate is subjected to ultracentrifugation for 40 to 70 minutes.

21. 21. The method of claim 20, wherein the filtrate is subjected to ultracentrifugation at a speed of 100,000 x g (RCF).

22. 22. The method of claim 21, wherein the filtrate is subjected to ultracentrifugation for 60 minutes.

23. 23. The method of claim 22, wherein the exosomes in the pellet have a diameter of about 40 to 120 nm.

24. Culturing hIDPSCs in a basal medium; replacing the basal medium with serum-free medium when the hIDPSCs reach at least 80% confluence; harvesting the serum-free medium after the hIDPSCs have been cultured in the serum-free medium for at least 12 hours; filtering the recovered serum-free medium to obtain a filtrate, thereby removing cells and cell debris from the recovered serum-free medium; and subjecting the filtrate to ultracentrifugation at a speed of 99,850-100,210 x g (RCF) at 3-6°C for 40-70 minutes to generate a pellet containing exosomes; and Optionally, resuspending the pellet; 14. The method of claim 13, consisting essentially of:

25. 25. The method of claim 24, wherein the basal medium is replaced with serum-free medium when the hIDPSCs are not more than 90% confluent.

26. 25. The method of claim 24, wherein the hIDPSCs are cultured in serum-free medium for no more than 48 hours, after which the serum-free medium is harvested.

27. 27. The method of any one of claims 24 to 26, wherein the cell culture medium is filtered using a 0.22 μm cellulose acetate membrane.

28. 28. The method of claim 27, wherein the exosomes in the pellet have a diameter of about 20 to 150 nm.

29. The method of any one of claims 13 to 17 and 24 to 26, wherein the pellet is free of polyethylene glycol.

30. 27. The method of any one of claims 1 to 5, 13 to 17, and 24 to 26, wherein ultracentrifugation is carried out in a centrifuge equipped with a swing-out rotor that holds centrifuge tubes with a capacity of at least 15 mL.

31. At least 1 x 10 per mL of filtrate １０ 27. The method of any one of claims 1 to 5, 13 to 17, and 24 to 26, wherein exosomes are isolated.

32. 27. The method of any one of claims 1 to 5, 13 to 17, and 24 to 26, wherein the pellet is resuspended in sterile saline (0.9%).

33. 27. The method of any one of claims 1 to 5, 13 to 17, and 24 to 26, wherein the exosomes express at least one marker selected from integrin, intercellular adhesion molecule 1 (ICAM-1), epithelial cell adhesion molecule (EpCAM), CD31, annexin, TSG01, apoptosis-related gene-interacting protein X (ALIX), Rab5b, HLA-G, HSP70, CD63, lysosomal-associated membrane protein 2 (LAMP2), and lysosomal integral membrane protein (LIMP).

34. 34. The method of claim 33, wherein at least one marker is CD63.

35. 27. The method of any one of claims 1 to 5, 13 to 17, and 24 to 26, wherein the exosomes comprise proteins, lipids, cytosolic components, and nucleic acids.

36. 37. The method of Claim 36, wherein the nucleic acids comprise miRNA and lncRNA.

37. 27. The method of any one of claims 1 to 5, 13 to 17, and 24 to 26, wherein the hIDPSCs are negative for CD11B, CD19, CD34, CD45, CD89, CD86, and HLA-DR.

38. 27. The method of any one of claims 1 to 5, 13 to 17, and 24 to 26, wherein the hIDPSCs are positive for CD73, CD90, CD105, and CD146.

39. A pharmaceutical composition comprising exosomes isolated according to the method of any one of claims 1 to 39 and resuspended in isotonic saline.

40. Exosomes isolated according to the method of any one of claims 1 to 39, and Pharmaceutically acceptable carriers or excipients A pharmaceutical composition comprising:

41. Exosomes isolated from human immature dental pulp stem cells (hIDPSCs), and Pharmaceutically acceptable carriers or excipients A pharmaceutical composition comprising:

42. 42. The pharmaceutical composition of any one of claims 39 to 41, formulated for local, intranasal, intravenous (IV) or intrathecal injection.

43. 42. A method for treating a neurological disease or condition, an infectious disease, or cancer, comprising administering to a subject a pharmaceutical composition according to any one of claims 39 to 41.

44. 44. The method of claim 43, wherein the pharmaceutical composition is administered to the subject intravenously.

45. 45. The method of claim 43 or 44, wherein the neurological disease or condition is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia, spinocerebellar ataxia, and Huntington's disease (HD).

46. 46. ​​The method of claim 45, wherein the neurological disease or condition is selected from ALS and HD.

47. 45. The method of claim 43 or 44, wherein the infectious disease is selected from the group comprising novel coronavirus disease (COVID-19), salmonella infection, dysentery, pathogenic Escherichia coli infection, human cytomegalovirus infection, Kaposi's sarcoma-associated herpesvirus infection, influenza infection, Legionnaires' disease, and malaria.

48. 48. The method of claim 47, wherein the infectious disease is COVID-19.

49. 45. The method of claim 43 or 44, wherein the cancer is selected from the group comprising hepatocellular carcinoma, colorectal cancer, endometrial cancer, thyroid cancer, and bladder cancer.

50. 40. A pharmaceutical composition comprising exosomes isolated according to the method of any one of claims 1 to 39 for use in the treatment of a neurological disease or condition, an infectious disease, or cancer.

51. 51. The pharmaceutical composition for use according to claim 50, wherein the neurological disease or condition is amyotrophic lateral sclerosis (ALS) or Huntington's disease (HD).

52. 51. The pharmaceutical composition for use according to claim 50, wherein the infectious disease is novel coronavirus disease (COVID-19).

53. 42. Use of the pharmaceutical composition of any one of claims 39 to 41 for the treatment of a neurological disease or condition, an infectious disease, or cancer.

54. 54. The use of claim 53, wherein the neurological disease or condition is amyotrophic lateral sclerosis (ALS) or Huntington's disease (HD).

55. The use according to claim 53, wherein the infectious disease is novel coronavirus disease (COVID-19).

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