Amniotic fluid-derived extracellular vesicles and uses thereof for wound healing

Abstract

To provide compositions and methods for wound healing in a subject.SOLUTION: Methods include administering a therapeutic amount of a pharmaceutical composition comprising extracellular vesicles from amniotic fluid and a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 812,011, filed February 28, 2019, the contents of which are incorporated by reference in their entirety.

[0002] The described invention generally relates to purified amniotic fluid-derived extracellular vesicles (EVs), compositions comprising EVs, and uses thereof. [Background technology]

[0003] Amniotic fluid components The fetal adnexa (meaning the connecting parts) consists of the placenta, the egg membrane, and the umbilical cord. At term, the placenta is disc-shaped with a diameter of 15-20 cm and a thickness of 2-3 cm. The amnion, amnion, and chorion surround the fetus within the amniotic cavity, and the decidua uteri extends from the edge of the chorionic disc. The chorionic plate (the fetal component of the extraembryonic tissue) is a multi-layered structure facing the amniotic cavity. It consists of two different structures: the amnion (consisting of the epithelium, stratum compactum, amniotic mesoderm, and spongiosum) and the chorion (consisting of a region of mesenchyme and extravillous proliferative trophoblast cells, which are inserted into a variable amount of Langhansoid fibrin that is covered or not by syncytiotrophoblasts).

[0004] The villi originate from the chorionic plate and anchor the placenta through the substrate trophoblast and maternal endometrium. From the maternal side, substrate projections within the chorionic villi generate placental septa, which divide the parenchyma into irregular cotyledons (Parolini, O. et al., 2008, Stem Cell, 2008, 26: 300-311).

[0005] Some villi anchor the placenta to the substrate, while others terminate freely in the intervillous space. Chorionic villi exhibit different functions and structures. In the full-term placenta, stem villi show an inner core of fetal blood vessels with a well-defined muscular wall and connective tissue composed of fibroblasts, myofibroblasts, and dispersed tissue macrophages (Hofbauer cells). Mature intermediate and full-term villi consist of capillaries and thin mesenchyme. A basement membrane separates the interstitial core from an uninterrupted multinucleated layer called syncytiotrophoblasts. Between the syncytiotrophoblasts and their basement membrane is a single or aggregated Langhans cytotrophoblast layer, commonly referred to as the cytotrophoblast (Parolini, O. et al., 2008, Stem Cell, 2008, 26: 300-311).

[0006] The placenta contains three layers: the amnion, which is derived from the embryo, the chorion, and the decidua, which is derived from maternal tissues. The chorion is derived from the trophoblast, and the amnion is derived from the epiblast, which gives rise to all germ layers of the embryo as early as 8 days after fertilization.

[0007] Four regions of the fetal placenta can be distinguished: the amniotic epithelial region, the amniotic mesenchymal region, the chorionic mesenchymal region, and the chorionic trophoblast region.

[0008] amnion The amnion is a thin avascular membrane consisting of an inner epithelial layer and an outer connective tissue layer, which is adjacent to the fetal skin on the umbilical cord. The outer layer contains human amniotic mesenchymal stromal cells (hMSCs) surrounded by an intracellular matrix (Grzywocz, Z. et al. Folia Histochemica et Cytobiologica (2014) 52(3):163-170). The inner layer closest to the fetus is the amniotic epithelium (AE), which is an uninterrupted single layer of flat cuboidal and columnar epithelial cells that is in contact with the amniotic fluid. It is attached to a distinct basement membrane that connects to the amniotic germ layer (AM). In the amniotic germ layer closest to the epithelium, an acellular lamina densa composed of collagens I and III and fibronectin is discernible. Deep to the AM, a network of dispersed fibroblast-like mesenchymal cells and rare macrophages is observed. The mesenchymal layer of the amnion has been reported to contain two subfractions, one containing a mesenchymal phenotype, also known as amniotic mesenchymal stem cells, and the other containing monocyte-like cells. The amnion lacks blood vessels and nerves. It obtains its nutrition directly by diffusion from the amniotic fluid.

[0009] Chorion A spongy layer of loosely arranged collagen fibers separates the amniotic and chorionic mesoderm. The chorion (chorion) consists of regions of mesoderm and trophoblast. The chorion and amniotic mesoderm are similar in composition. A large, incomplete basement membrane separates the chorionic mesoderm from the extravillous trophoblast cells. The latter, like the trophoblast cells present in the substrate, are dispersed within the fibrinoid layer and express immunohistochemical markers of proliferation. The Langhans fibrinoid layer normally increases during pregnancy and consists of two different types of fibrinoid: an inner matrix type (more compact) and an outer fibrin type (more reticular). At the edge and base of the placenta, the trophoblast is extensively interdigitated with the decidua (Cunningham, F. et al., The placenta and fetal membranes, Williams Obstetrics, 20th ed. Appleton and Lange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of the human placenta. New York, Springer-Verlag, 2000, 42-46, 116, 281-297).

[0010] Amniotic membrane-derived stem cells The amniotic membrane itself contains pluripotent cells that can differentiate in various layers. Studies have reported their potential in neural and glial cells, cardiac repair, and even hepatic cells. Studies have shown that human amniotic epithelial cells express stem cell markers and have the ability to differentiate into all three germ layers. These properties, the ease of cell isolation, and the availability of the placenta make the amniotic membrane a useful and undisputed source of cells for transplantation and regenerative medicine.

[0011] Amniotic epithelial cells can be isolated from the amniotic membrane by several methods known in the art. According to one such method, the amniotic membrane is peeled from the underlying chorion and digested with trypsin or other digestive enzymes. The isolated cells readily attach to plastic or basement membrane-coated culture dishes. Cultures are typically established in simple media such as Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5%-10% serum and epidermal growth factor (EGF), in which the cells grow robustly and exhibit a typical cuboidal epithelial morphology. Two to six passages are usually possible before proliferation ceases. Amniotic epithelial cells do not grow well at low density.

[0012] Amniotic membrane contains epithelial cells with different surface markers, suggesting phenotypic heterogeneity. Immediately after isolation, human amniotic epithelial cells express very low levels of human leukocyte antigen (HLA)-A, B, C; however, by the second passage, significant levels are observed. Additional cell surface antigens on human amniotic epithelial cells include, but are not limited to, ATP-binding cassette transporter G2 (ABCG2 / BCRP), CD9, CD24, E-cadherin, integrin α6 and 1, c-met (hepatocyte growth factor receptor), stage-specific embryonic antigen (SSEA) 3 and 4, and tumor rejection antigens 1-60 and 1-81. Surface markers that appear to be absent on human amniotic epithelial cells include SSEA-1, CD34, and CD133, while other markers such as CD117 (c-kit) and CCR4 (CC chemokine receptor) may be negative or expressed at very low levels on some cells. Initial cell isolates express very low levels of CD90 (Thy-1), but expression of this antigen increases rapidly during culture (Miki, T. et al., Stem Cells, 2005, 23:1549-1559; Miki, T. et al., Stem Cells, 2006, 2:133-142).

[0013] In addition to surface markers, human amniotic epithelial cells express molecular markers of pluripotent stem cells, such as octamer-binding protein 4 (OCT-4), SRY-related high-molecular-weight box gene 2 (SOX-2), and Nanog (Miki, T. et al., Stem Cells, 2005, 23:1549-1559).

[0014] Human amniotic mesenchymal cells (hAMSCs) and human chorionic mesenchymal cells (hCMSCs) are believed to originate from the extraembryonic mesoderm. hAMSCs and hCMSCs can be isolated from amniotic and chorionic mesoderm, stage I, stage II, and stage III mesoderm, respectively. In the case of hAMSCs, isolation is usually performed using amniotic membranes dissected from the flexed part of the egg membrane to minimize the presence of maternal cells. For example, homogenous hAMSC populations can be obtained in a two-step procedure; whereby minced amniotic tissue is treated with trypsin to remove hAECs, and the remaining mesenchymal cells are liberated by digestion (e.g., with collagenase or collagenase and DNase). The yield from term amniotic membrane is approximately 1 million hAMSCs and 10 times more hAECs per gram of tissue (Casey, M. and MacDonald P., Biol Reprod, 1996, 55:1253-1260).

[0015] hCMSCs are isolated from both first and third stage chorions after mechanical and enzymatic removal of the trophoblast layer with dispase. The chorionic mesoderm tissue is then digested (e.g., with collagenase or with collagenase and DNase). Mesenchymal cells have also been isolated from chorionic fetal villi by explant culture, but this is likely due to maternal contamination (Zhang, X., et al., Biochem Biophys Res Commun, 2006, 340: 944-952; Soncini, M. et al., J Tissue Eng Regen Med, 2007, 1: 296-305; Zhang et al., Biochem Biophys Res Commun, 2006, 351: 853-859).

[0016] The surface marker characteristics of cultured hAMSCs and hCMSCs, as well as mesenchymal stromal cells (MSCs) from adult bone marrow, are similar.

[0017] Amniotic fluid (AF) Amniotic fluid is a complex and dynamic biological fluid that provides mechanical protection, nutrients, and other molecules necessary for fetal growth and health (Cho, CK.J., et al, “Proteomics Analysis of Human Amniotic Fluid,” 2007 Molecular & Cellular Proteomics 6:1406-1415). The quantitative and qualitative integrity of AF is essential for normal development of the human fetus during pregnancy.

[0018] During embryonic development, the amniotic cavity first appears 7-8 days after fertilization, and in early pregnancy, amniotic fluid is derived primarily from maternal plasma passing through the egg membrane (Rennie, K. et al., “Applications of amniotic membrane and fluid in stem cell biology and regenerative medicine,” Stem Cells Intl. (2012) article 721538). Fetal urine first enters the amniotic space at 8-11 weeks of gestation, and in the second half of pregnancy, fetal urine becomes the main source of amniotic fluid (ibid.). At this time, keratinization of the fetal skin is complete, reducing water transport across the skin and decreasing AF osmolality (ibid.). For the remainder of pregnancy, fluid volume is determined by a variety of mechanisms, including fetal urine production, oral, nasal, tracheal and pulmonary fluid secretion, fetal swallowing, and contributions from intramembranous pathways (ibid.).

[0019] AF contains water, amino acids, peptides, proteins, carbohydrates, lipids, lactate, pyruvate, enzymes, growth factors, hormones, and electrolytes (Cho, CK. J., et al, “Proteomics Analysis of Human Amniotic Fluid,” Molecular & Cellular Proteomics (2007) 6: 1406-1415; Rennie, K. et al., “Applications of amniotic membrane and fluid in stem cell biology and regenerative medicine,” Stem Cells Intl. (2012) article 721538). Although the main component of AF is water, its overall composition varies throughout pregnancy (Roubelakis,MG,et al.,“Amniotic fluid and amniotic membrane stem cells: marker discovery,'Stem Cells Intl(2012)article 107836). Furthermore, fluid secretions from the fetus into the AF carry a variety of fetal cells, resulting in a heterogeneous population of cells originating from the fetal skin, gastrointestinal, respiratory, urinary tract, and amniotic membrane (Rennie,K.et al.,“Applications of amniotic membrane and fluid in stem cell biology and regenerative medicine,” Stem Cells Intl.(2012)article 721538). As the fetus develops, the volume and composition of amniotic fluid changes significantly, and the complement of cells detected in amniotic fluid samples taken at various gestational ages varies considerably.

[0020] Amniotic fluid cells (AFCs) represent a heterogeneous population derived from three germ layers. These cells are of epithelial origin and originate either from the developing embryo or from the inner surface of the amniotic membrane, characterized as amniotic stem cells (Roubelakis, MG, et al., “Amniotic fluid and amniotic membrane stem cells: marker discovery,” Stem Cells Intl (2012) article 107836). AFCs mainly consist of three groups of adherent cells classified based on morphological, growth, and biochemical characteristics: epithelial-like (E-type) cells, which are cuboidal to columnar cells derived from fetal skin and urine; amniotic fluid (AF-type) cells derived from the fetal membrane; and fibroblastic (F-type) cells, which are mainly derived from fibrous connective tissue. The predominant cell type appears to be the AF-type, which co-expresses keratin and vimentin. Several studies have reported that human amniotic fluid stem cells (AFSCs) can be obtained from small amounts of second-trimester AFs retrieved during routine amniocentesis. The isolation of AFSCs can be classified as follows: (i) a single-step culture protocol in which primary cultures are left stationary for more than 7 days until the first colonies appear; (ii) a two-step culture protocol in which non-adherent amniotic cells are harvested and further expanded after 5 days of culture; (iii) selection for the cell surface marker CD117 (c-kit receptor); (iv) mechanical isolation of primary mesenchymal progenitor colonies formed in the initial culture; and (v) short-term culture to isolate fibroblast-like colonies. The majority of AFSCs isolated following these steps shared a multipotent mesenchymal phenotype and showed high proliferation and broad differentiation potential compared to adult MSCs (Roubelakis,MG,et al.,“Amniotic fluid and amniotic membrane stem cells:marker discovery,” Stem Cells Intl(2012)article 107836).

[0021] Detailed analysis of AFSC-conditioned medium revealed the presence of pro- and anti-angiogenic factors using Liminex's MAP technology. Vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and two angiogenesis inhibitors, interferon gamma (IFNγ) and interferon gamma-inducible protein 10 (IP-10), have been identified as secreted proteins (Id.). Relatively small numbers of AFSCs were shown to be sufficient to secrete detectable amounts of pro-angiogenic growth factors and cytokines (Id.).

[0022] Human amniotic fluid proteome Analysis of human AF samples from women aged 16-18 weeks showed that albumin constituted nearly 70% of the protein content of AF, with immunoglobulins being the second most abundant fraction (Cho, CK.J., et al., “Proteomics Analysis of Human Amniotic Fluid,” 2007, Molecular & Cellular Proteomics 6:1406-1415). Cho et al. identified 842 proteins from 754 distinct genes and 88 proteins from uncharacterized genes in amniotic fluid. Proteins were sorted by the number of unique peptides identified from strong anion exchange (SAX) and strong cation exchange (SCX) methods, which are generally accepted as semiquantitative measures of protein abundance. The top 15 proteins in amniotic fluid with the highest number of unique peptides were, in descending order, albumin, immunoglobulins, fibronectin, serotransferrin, complement C3, α1-antitrypsin, ceruloplasmin, α-fetoprotein, vitamin D binding protein, periostin, apolipoprotein A-1, antithrombin III, transforming growth factor-β-induced protein ig-h3 precursor, α1-microglobulin, and plasminogen. In comparison, the top 15 proteins in plasma were, in descending order, albumin, immunoglobulins, serotransferrin, fibrinogen, α1-microglobulin, α1-antitrypsin, complement C3, haptoglobin, apolipoprotein A-1, apolipoprotein B, α1-acid glycoprotein, lipoprotein, factor H, ceruloplasmin, and complement C4.

[0023] Metabolomics Standard biochemical variables were measured in pure samples of amniotic fluid and extraembryonic coelomic fluid obtained by transvaginal ultrasound-guided amniocentesis from women with normal pregnancies of 7-12 weeks' gestation that had been terminated. During the first trimester of pregnancy, sodium, potassium, and bicarbonate levels were significantly higher in amniotic fluid, whereas chloride, urea, bilirubin, protein, albumin, glucose, creatinine, calcium, and phosphate were present in higher concentrations in the extraembryonic fluid (Campbell, J. et al., “Biochemical composition of amniotic fluid and extrambryonic coelomic fluid in the first trimester of pregnancy,” Br. J. Obstet. Gynaecol. (1992) 99(7):563-565).

[0024] 1H-NMR-based metabolic signature was applied to track metabolic changes occurring in amniotic fluid and plasma of healthy mothers during pregnancy (Orczyk-Pawilowicz, et al, “Metabolomics of human amniotic fluid and maternal plasma during normal pregnancy,” PLos ONE (2016) 11(4): e0152740). During the first two-thirds of pregnancy, it is established that the mother is in an anabolic state. During the third trimester, intensive anabolic activity occurs in the fetus, and maternal metabolism switches to catabolic. In AF, the transition from the second to the third trimester was associated with decreases in the levels of glucose, carnitine, and amino acids (valine, leucine, isoleucine, alanine, methionine, tyrosine, and phenylalanine) and increases in the levels of creatinine, succinate, pyruvate, choline, N,N-dimethylglycine, and urocanic acid. In plasma, the progression from the second to the third trimester was associated with increased levels of glycerol, choline, and ketone bodies (3-hydroxybutyrate and acetoacetate), whereas pyruvate concentrations significantly decreased. The lactate to pyruvate ratio decreased in AF and increased in plasma. The researchers concluded that these results are most likely related to changes in fetal growth dynamics, i.e., the transition to a fast weight gain phase that requires a significantly higher rate of anabolic processes. The significant decrease in amino acid levels in AF, in contrast to plasma, may be related to fetal maturation and the increased demand for basic building blocks necessary for protein synthesis.

[0025] Amniotic fluid and tissue contain numerous active biomolecules, including proteins, lipids, carbohydrates, and electrolytes; some of which may function as enzymes, hormones, and growth factors. Growth factors are usually proteins that can have diverse biological effects, but are characterized as trophic factors that can activate growth-promoting cellular signaling cascades. Some biologically relevant growth factors found in amniotic fluid include epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), insulin-like growth factor (IGF), and erythropoietin (EPO). Amniotic fluid also reduces scarring (Ozgenel GY et al., J Neurosurg 2003;98:371-377), in part due to the presence of hyaluronic acid (Gao X et al., Ann Plastic Surg 1994;33:128-134).

[0026] Amniotic fluid growth factor activity One of the functions of amniotic cells is the release of growth factors and cytokines that regulate various processes during embryonic development (Grzywocz,Z.et al. Folia Histochemica et Cytobiologica(2014)52(3):163-170). During fetal development, VEGF increases the permeability of the human amniotic membrane. In vitro studies have shown that growth factors produced by the amniotic membrane are involved in angiogenesis, re-epithelialization, and immune regulation. Some factors, e.g., macrophage colony-stimulating factor (M-CSF), stimulate cell differentiation and proliferation. Other factors, such as IGF-2, can promote proliferation.

[0027] Growth factors produced by amniotic cell fractions and whole amniotic tissue were measured using an in vitro cytokine assay (ibid.). The assays revealed that the supernatant contained epidermal and fibroblast growth factors (HB-EGF, EGF-2, EGF-R, bFGF, FGF-4, FGF-6, FGF-7), nerve and glial growth factors (bNGF, GDNF, NT-3, NT-4), angiogenic growth factors (VEGF, VEGF-D, VEGF-R2, VEGF-R3, PLGF), hematopoietic growth factors (G-CSG, GM-CSF, M-CSF, M-CSF-R, SCF, SCF- R), insulin-like growth factors (IFG-1, IGF-2, IGF-ISR, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6), platelet-derived growth factors (PDGF-AA, PDGF-AB, PDGF-BB, PDGFRa, PDGFRb), transforming growth factors (TGF-a, TGF-b, TGF-b2, TGF-b3) and other proteins (HGF, AR) were detected (Id.). The study focused on statistically significant time-dependent changes in the levels of growth factors and their receptors measured at 3, 6, 24, and 48 hours (Id.). Cell fractions were isolated as described by Soncini et al. with minor modifications (J. Tissue Eng. Regen. Med. (2007) 1:296-305). Whole human amniotic membranes, consisting of equal amounts of hAMSCs and hAECs, released EGF-R, IGF-2, IGFBP-2, IGFBP-2, and IGFBP6 into the conditioned medium. Amniotic cell fraction 1, which stained positively for mesenchymal cell markers CD73 (86%), CD90 (19.3%), and CD105 (2.2%), released only NT-4, the concentration of which increased statistically over the study period, suggesting that NT4 played a local role in the function of the amniotic epithelium, possibly in relation to apoptosis (Id.). Amniotic cell fraction 2, which stained positively for epithelial cell markers cytokeratin 4 / 5 / 6 / 8 / 10 / 13 / 18 and contained mainly amniotic epithelial cells, released hematopoietic growth factors such as G-CSF, M-CSF, and PDGF, and the angiogenesis regulator PLGF into the conditioned medium (Id.).

[0028] Thus, amniotic tissue and fluid are a source of biological components that stimulate tissue repair and promote skin and connective tissue homeostasis. However, there is significant variability in the molecular composition of amniotic tissue and fluid from donor to donor. Furthermore, it is unclear whether many important amniotic factors survive the various processes used in amniotic fluid collection and storage. Thus, the inherent variability of amniotic tissue and the various collection and storage conditions pose a challenge to standardize and reproduce the efficacy of these products in various therapeutic applications.

[0029] wound healing Wounds result from injury or disruption of normal anatomical structure and function (Robson MC et al., Curr Probl Surg 2001, 38:72-140; Velnar T et al., The Journal of International Medical Research 2009, 37:1528-1542). This can range from simple disruption of the epithelial integrity of the skin to deeper subcutaneous tissues with damage to other structures such as tendons, muscles, blood vessels, nerves, parenchymal organs, and even bone (Alonso JE et al., Surg Clin North Am 1996, 76:879-903). Regardless of cause or form, wounds damage and disrupt the local tissue environment.

[0030] Wound healing is a dynamic, interactive process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells. The wound repair process can be divided into four (4) temporally and spatially overlapping phases: (1) the coagulation phase, (2) the inflammatory phase, (3) the proliferation phase, and (4) the remodeling phase. Much of what is known is based on human cutaneous wound healing.

[0031] Solidification stage Immediately after injury, platelets adhere to damaged blood vessels and initiate a release response, triggering the blood coagulation cascade that initiates a hemostatic response, prevents excessive bleeding, and temporarily protects the injured area. Platelets release well over a dozen growth factors, cytokines, and other survival or apoptosis inducers (Weyrich AS and Zimmerman GA, Trends Immunol 2004 Sep,25(9):489-495). Key components of the platelet release response include platelet-derived growth factor (PDGF) and transforming growth factors A1 and 2 (TGF-A1 and TGF-2), which attract inflammatory cells such as leukocytes, neutrophils, and macrophages (Singer AF and Clark RA, N Engl J Med 1999 Sep 2,341(10):738-746).

[0032] Inflammation stage Tissue injury leads to the destruction of blood vessels and the extravasation of blood components. The thrombus re-establishes hemostasis and provides a provisional extracellular matrix for cell migration. Platelets secrete several mediators of wound healing, such as platelet-derived growth factors, which not only promote the formation of a hemostatic plug but also attract and activate macrophages and fibroblasts (Heldin, C. and Westermark B., In: Clark R., ed. The molecular and cellular biology of wound repair, 2nd Ed. New York, Plenum Press, pp. 249-273, (1996)). However, in the absence of bleeding, it has been suggested that platelets are not essential for wound healing; a number of vasoactive mediators and chemotactic factors have been shown to be produced by the coagulation and activated complement pathways, as well as by injured or activated parenchymal cells, which recruit inflammatory leukocytes to the site of injury (ibid.).

[0033] Cellular invasion into the wound and activation of local cells are initiated by mediators either released de novo by resident cells or derived from reserves stored in the granules of platelets and basophils (Sephel, GC and Woodward, SC, 3. Repair, Regeneration and Fibrosis,” in Rubin's Pathology, Rubin, R. and Strayer, DS Eds;5 th Ed., Wolters Kluwyer Health, / Lippincott Williams & Wilkins, Philadelphia, PA (2008), at 71. Cell migration employs the response of cells to cytokines and the insoluble substrates of the extracellular matrix (Ibid., at 72).

[0034] Infiltrating neutrophils cleanse the wounded area of ​​foreign particles and bacteria, pushing them out in eschar (dead tissue that is either sloughed (sloughed) off from healthy skin or phagocytosed by macrophages). In response to specific chemoattractants, such as fragments of extracellular matrix proteins, transforming growth factor beta (TGF-β), and monocyte chemotactic protein-1 (MCP-1), monocytes also infiltrate the wound site and become activated macrophages that release growth factors (such as platelet-derived growth factor and vascular endothelial growth factor) that initiate the formation of granulation tissue. Macrophages bind to specific proteins of the extracellular matrix by integrin receptors; this is an action that stimulates the phagocytosis of microorganisms and extracellular matrix fragments by macrophages (Brown, E. Phagocytosis, Bioessays, 17:109-117 (1995)). Studies have also reported that adhesion to extracellular matrix stimulates monocytes to transform into inflammatory or reparative macrophages. These macrophages play a key role in the transition between inflammation and repair (Riches, D., In Clark R., Ed. The molecular and cellular biology of wound repair, 2nd Ed. New York, Plenum Press, pp.95-141). For example, adhesion induces the expression by monocytes and macrophages of colony-stimulating factor-1 (CSF-1), a cytokine required for monocyte and macrophage survival; tumor necrosis factor-α (TNF-α), a potent proinflammatory cytokine; and platelet-derived growth factor (PDGF), a potent chemoattractant and mitogen for fibroblasts. Other cytokines that have been shown to be expressed by monocytes and macrophages include transforming growth factor (TGF-α), interleukin-1 (IL-1), transforming growth factor β (TGF-β), and insulin-like growth factor-I (IGF-I) (Rappolee, D. et al., Science, 241, pp. 708-712 (1988)).Since animals depleted of macrophages have defects in wound repair, it has been suggested that monocyte- and macrophage-derived growth factors are required for the initiation and propagation of new tissue formation in wounds (Leibovich, S, and Ross, R., Am J Pathol, 78, pp 1-100 (1975)).

[0035] Multiplication stage The inflammatory phase is followed by a proliferative phase, in which active angiogenesis creates new capillaries and allows the delivery of nutrients to the wound site, especially to support fibroblast proliferation. Fibroblasts present in the granulation tissue are activated and acquire a smooth muscle cell-like phenotype. Differentiation of fibroblasts into myofibroblasts begins with the appearance of protomyofibroblasts, whose stress fibers contain only β- and γ-cytoplasmic actin. Protomyofibroblasts can evolve into differentiated myofibroblasts, whose stress fibers contain α-smooth muscle actin. Myofibroblasts synthesize and deposit extracellular matrix (ECM) components that replace the provisional matrix. They also have contractile properties mediated by α-smooth muscle actin organized into microfilament bundles or stress fibers.

[0036] angiogenesis To maintain the newly formed granulation tissue, new blood vessel formation (angiogenesis) is required. Angiogenesis is a complex process that depends on the extracellular matrix of the wound bed, as well as endothelial cell migration and mitogenic stimulation (Madri, J. et al., Angiogenesis in Clark, R. Ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, pp. 355-371 (1996)). Induction of angiogenesis was initially attributed to acidic or basic fibroblast growth factors. Many other molecules, such as vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), angiogenin, angiotropin, angiopoietin-1, and thrombospondin, have since been found to have angiogenic activity (Folkman, J. and D'Amore, P, Cell, 87, pp. 1153-1155 (1996)).

[0037] Low oxygen tension and elevated lactate have also been suggested to stimulate angiogenesis. These molecules induce angiogenesis by stimulating the production of basic fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) by macrophages and endothelial cells. For example, it has been reported that activated epidermal cells in wounds secrete large amounts of vascular endothelial growth factor (VEGF) (Brown, L. et al., J Exp Med, 176, 1375-1379 (1992)).

[0038] Basic fibroblast growth factor has been hypothesized to set the stage for angiogenesis during the first 3 days of wound repair, while vascular endothelial growth factor is important for angiogenesis during the formation of granulation tissue from days 4 to 7 (Nissen, N. et al., Am J Pathol, 152, 1445-1552 (1998)).

[0039] In addition to angiogenic factors, it has been shown that an appropriate extracellular matrix and endothelial receptors for provisional matrix are necessary for angiogenesis. Microvascular endothelial cells adjacent to and growing within a wound transiently deposit large amounts of fibronectin in the vessel wall (Clark, R. et al., J. Exp Med, 156, 646-651 (1982)). Because angiogenesis requires the expression of functional fibronectin receptors by endothelial cells (Brooks, P. et al., Science, 264, 569-571 (1994)), it has been suggested that perivascular fibronectin serves as a conduit for the migration of endothelial cells into the wound. Furthermore, it has been shown that the expression and activity of proteases are also necessary for angiogenesis (Pintucci, G. et al., Semin Thromb Hemost, 22, 517-524 (1996)).

[0040] The sequence of events leading to angiogenesis has been proposed as follows: Injury causes tissue destruction and hypoxia. Angiogenic factors, such as acidic and basic fibroblast growth factors (FGFs), are released by macrophages soon after cell destruction, and hypoxia stimulates the production of vascular endothelial growth factor by epidermal cells. Proteolytic enzymes released into connective tissues degrade extracellular matrix proteins. Fragments of these proteins recruit peripheral blood monocytes to the site of injury, where they become activated macrophages and release angiogenic factors. Specific macrophage angiogenic factors, such as basic fibroblast growth factor (bFGF), stimulate endothelial cells to release plasminogen activators and procollagenase enzymes. Plasminogen activators convert plasminogen to plasmin and procollagenase to active collagenase, and these two proteases work together to digest the basement membrane. Fragmentation of the basement membrane allows endothelial cells stimulated by angiogenic factors to migrate and form new blood vessels at the site of injury. Once the wound is filled with new granulation tissue, angiogenesis ceases and many of the new blood vessels collapse as a result of apoptosis (Ilan, N. et al., J Cell Sci, 111, 3621-3631 (1998)). It has been suggested that this programmed cell death is regulated by various matrix molecules such as thrombospondin 1 and 2, and antiangiogenic factors such as angiostatin, endostatin, and angiopoietin 2 (Folkman, J., Angiogenesis and angiogenesis inhibition: an overview, EXS, 79, 1-8, (1997)).

[0041] Remodeling Phase The fourth healing phase involves the gradual remodeling and re-epithelialization of the granulation tissue. This remodeling process is mainly mediated by proteolytic enzymes, especially matrix metalloproteinases (MMPs) and their inhibitors (TIMPs, tissue inhibitors of metalloproteinases). During re-epithelialization, type III collagen, the main component of granulation tissue, is gradually replaced by type I collagen, the main structural component of the dermis. Elastin, which contributes to skin elasticity and is absent in granulation tissue, also reappears. Cell density normalizes through apoptosis (resolution) of vascular cells and myofibroblasts.

[0042] epithelialization Wound re-epithelialization begins within hours after injury. Epidermal cells from skin appendages such as hair follicles rapidly clear clotted blood and damaged stroma from the wound space. At the same time, cells undergo phenotypic changes including contraction of intracellular tonofilaments (Paladini, R. et al., J. Cell Biol, 132, pp. 381-397 (1996)); dissolution of most intercellular desmosomes that provide physical connections between cells; and formation of peripheral cytoplasmic actin filaments that allow cell movement and migration (Goliger, J. and Paul, D. Mol Biol Cell, 6, pp. 1491-1501 (1995); Gabbiani, G. et al., J Cell Biol, 76, PP. 561-568 (1978)). In addition, epidermal and dermal cells no longer adhere to each other because hemidesmosome junctions between the epidermis and basement membrane dissolve, allowing lateral movement of epidermal cells. Expression of integrin receptors on epidermal cells allows them to interact with various extracellular matrix proteins (e.g., fibronectin and vitronectin) that are interspersed with interstitial type I collagen at the wound edge and interwoven with the fibrin clot in the wound space (Clark, R., J Invest Dermatol, 94, Suppl, pp. 128S-134S (1990)). Migrating epidermal cells deconstruct the wound, separating the dry eschar from viable tissue. The path of deconstruction appears to be determined by the array of integrins that migrating epidermal cells express on their cell membranes.

[0043] The degradation of the extracellular matrix required for epidermal cells to migrate between the collagenous dermis and the fibrin eschar depends on the production of collagenase by epidermal cells (Pilcher, B. et al., J Cell Biol, 137, pp. 1445-1457 (1997)) and on the activation of plasmin by plasminogen activators produced by dermal cells (Bugge, T. et al., Cell, 87: 709-719 (1996)). Plasminogen activators also activate collagenase (matrix metalloproteinase-1) (Mignatti, P. et al., Proteinases and Tissue Remodeling. In Clark, R. Ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, 427-474 (1996)), promoting the degradation of collagen and extracellular matrix proteins.

[0044] One to two days after injury, epidermal cells at the wound edge begin to proliferate behind actively migrating cells. The stimuli for epidermal cell migration and proliferation during re-epithelialization remain to be determined, but several possibilities have been suggested. The absence of cells adjacent to the wound edge (the "free edge" effect) may indicate both migration and proliferation of epidermal cells. Local release of growth factors and increased expression of growth factor receptors may also stimulate these processes. The main candidates include epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor (KGF) (Nanney, L. and King, L. Epidermal Growth Factor and Transforming Growth Factor-α. In Clark, R. Ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, pp. 171-194 (1996); Werner, S. et al., Science, 266, pp. 819-822 (1994); Abraham, J. and Klagsburn, M. Modulation of Wound Repair by Members of the Fiborblast Growth Factor family. In Clark, R. Ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, pp. 195-248 (1996)). As re-epithelialization continues, basement membrane proteins reappear in a highly ordered, zipper-like fashion from the wound edge inwards (Clark R. et al., J. Invest Dermatol, 79, pp. 264-269 (1982)). Epidermal cells revert to their normal phenotype and once again firmly attach to the established basement membrane and underlying dermis.

[0045] Granulation tissue formation New stroma, often called granulation tissue, begins to invade the wound space about four days after injury. Numerous new capillaries give the new stroma its granular appearance. Macrophages, fibroblasts, and blood vessels migrate simultaneously into the wound space (Hunt, T. ed. Wound Healing and Wound Infection: Theory and Surgical Practice. New York, Appleton-Century-Crofts (1980)). Macrophages provide a continuous source of growth factors necessary to stimulate fibroplasia and angiogenesis; fibroblasts generate new extracellular matrix necessary to support cellular ingrowth; and blood vessels transport oxygen and nutrients necessary to maintain cellular metabolism.

[0046] Growth factors, particularly platelet-derived growth factor-4 (PDGF-4) and transforming growth factor beta-1 (TGF-β1) (Roberts, A. and Sporn, M, Transforming Growth Factor-1, In Clark, R. ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, pp. 275-308 (1996)), have been shown to stimulate fibroblasts in the tissue surrounding the wound to proliferate, express appropriate integrin receptors, and migrate into the wound space in cooperation with extracellular matrix molecules (Gray, A. et al., J Cell Sci, 104, pp. 409-413 (1993); Xu, J. and Clark, R., J Cell Biol, 132, pp. 239-149 (1996)). It has been reported that platelet-derived growth factor promotes the healing of chronic pressure ulcers (Robson, M. et al., Lancet, 339, pp. 23-25 ​​(1992) and diabetic ulcers (Steed, D., J Vasc Surg, 21, pp. 71-78 (1995)). In some other cases, basic fibroblast growth factor (bFGF) was effective in treating chronic pressure ulcers (Robson, M. et al., Ann Surg, 216, pp. 401-406 (1992)).

[0047] Structural molecules of the newly formed extracellular matrix, called the provisional matrix (Clark, R. et al., J. Invest Dermatol, 79, pp. 264-269, 1982), contribute to the formation of granulation tissue by providing a scaffold or conduit for cell migration. These molecules include fibrin, fibronectin, and hyaluronic acid (Greiling, D. and Clark R., J. Cell Sci, 110, pp. 861-870 (1997)). It has been suggested that the appearance of fibronectin and appropriate integrin receptors on fibroblasts that bind to fibronectin, fibrin, or both, is a rate-limiting step in the formation of granulation tissue. Fibroblasts are involved in the synthesis, deposition, and remodeling of the extracellular matrix, which itself can positively or negatively affect the ability of fibroblasts to perform these tasks and generally interact with their environment (Xu, J. and Clark, R., J Cell Sci, 132, pp. 239-249 (1996); Clark, R. et al., J Cell Sci, 108, pp. 1251-1261).

[0048] Migration of cells into cross-linked fibrin clots or tightly woven extracellular matrices requires an active proteolytic system capable of cleaving the pathways of cell migration. In addition to serum-derived plasmin, various fibroblast-derived enzymes, such as plasminogen activators, collagenases, gelatinase A, and stromelysin, have been suggested as potential candidates for this task (Mignatti, P. et al., Proteinases and Tissue Remodeling. In Clark, R. Ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, 427-474 (1996); Vaalamo, M. et al., J Invest Dermatol, 109, pp. 96-101 (1997)). After migrating into the wound, fibroblasts begin to synthesize the extracellular matrix. The provisional extracellular matrix is ​​gradually replaced by a collagen matrix, presumably in response to transforming growth factor-β1 (TGF-β1) signaling (Clark, R. et al., J Cell Sci, 108, pp. 1251-1261 (1995); Welch, M. et al., J. Cell Biol, 110, pp. 133-145 (1990)).

[0049] Once a rich collagen matrix is ​​deposited in the wound, fibroblasts cease producing collagen and the fibroblast-rich granulation tissue is replaced by a relatively acellular scar. Cells within the wound undergo apoptosis triggered by unknown signals. Dysregulation of these processes has been reported to occur in keloid formation, hypertrophic scars, dysmorphic scars, and fibrotic diseases such as scleroderma.

[0050] Wound contraction and extracellular matrix reorganization Wound contraction involves a complex and orchestrated interplay of cells, extracellular matrix, and cytokines. During the second week of healing, fibroblasts adopt a myofibroblast phenotype characterized by large bundles of actin-containing microfilaments arranged along the cytoplasmic face of the plasma membrane of cells by cell-cell and cell-matrix junctions (Welch, M. et al., J Cell Biol, 110, 133-145 (1990); Desmouliere, A. and Gabbiani, G. The role of the myofibroblast in wound healing and fibrocontractive diseases. In Clark, R. Ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, pp. 391-423 (1996)). The appearance of myofibroblasts corresponds to the onset of connective tissue compression and wound contraction. It has been suggested that this contraction requires stimulation with transforming growth factor (TGF)-β1 or β2 and platelet-derived growth factor (PDGF), adhesion of fibroblasts to the collagen matrix via integrin receptors, and cross-linking between individual collagen bundles (Montesano, R. and Orci, Proc Natl Acad Sci USA, 85, 4894-4897 (1988); Clark, R. et al., J Clin Invest, 84, 1036-1040 (1989); Schiro, J. et al., Cell, 67, 403-410 (1991); Woodley, D. et al., J Invest Dermatol, 97, 580-585 (1991)).

[0051] Collagen remodeling during the transition from granulation tissue to scar depends on the continued synthesis and catabolism of collagen at a low rate. Collagen degradation in wounds is controlled by several proteolytic enzymes called matrix metalloproteinases (MMPs), which are secreted by macrophages, epidermal cells, endothelial cells, and fibroblasts (Mignatti, P. et al., Proteinases and Tissue Remodeling. In Clark, R. Ed. The molecular and cellular biology of wound repair. 2nd Ed. New York, Plenum Press, 427-474 (1996)). It has been suggested that the various stages of wound repair depend on different combinations of matrix metalloproteinases and tissue inhibitors of metalloproteinases (Madlener, M. et al., Exp Cell Res, 242, 201-210 (1998)).

[0052] Wounds gained only about 20% of their ultimate strength in the first 3 weeks, during which time fibrillar collagen accumulated relatively rapidly and was remodeled by wound contraction. Thereafter, wounds gained tensile strength at a slower rate, reflecting a much slower rate of collagen accumulation and collagen remodeling with the formation of larger collagen bundles and an increased number of intermolecular crosslinks.

[0053] Signaling pathways involved in wound healing Wound healing is a complex process of cell proliferation, migration, matrix synthesis and contraction, involving various types of cells and regulatory mechanisms. Resident cells (keratinocytes, fibroblasts, and endothelial cells) and inflammatory cells are involved in wound healing (Song, Q, et al. Int J Mol Med. 2017 Aug; 40 (2): 465-473, Chen XH, et al. Int J Radiat Biol. 2009; 85: 607-613; Amadeu TP, et al. J Surg Res. 2008; 149: 84-9). Evidence has revealed that several signaling pathways, such as Janus-activated kinase / signal transducer and activator of transcription 3 (JAK / STAT3) signaling, are associated with wound healing through induction of their target gene expression (ibid. citing Li PN,et al. Wound Repair Regen. 2015;23:65-73; Pakyari M,et al. Adv Wound Care(New Rochelle)2013;2:215-224; Ren X,et al. Lasers Med Sci.2016;31:673-678; Shi Y,et al. Stem Cell Res Ther. 2015;6:120). In wound healing, cytokines contribute to the activation of STATs, and the activated JAK / STAT3 pathway controls proliferation and differentiation required for wound healing (ibid., Tokumaru S,et al. Biochem Biophys Res Commun. 2005;327:100-105;Yasukawa H,et al. Nat Immunol. 2003;4:551-556). Furthermore, through activation of the JAK / STAT3 signaling cascade, cytokines induce anti-apoptotic pathways and antimicrobial molecules to prevent tissue damage and assist in their repair (ibid., Lejeune D,et al. J Biol Chem. 2002;277:33676-33682;Wolk K,et al. Semin Immunopathol. 2010;32:17-31;Yu R,et al. Arch Oral Biol. 2016;72:14-20).Furthermore, one study showed that STAT3 played an important role in keratinocyte migration, but not proliferation, in wound healing (ibid., citing Sano S,et al. EMBO J. 1999;18:4657-4668). The crucial role of Smad3 signaling in cutaneous wound healing has been well documented (ibid., citing Li PN,et al. Wound Repair Regen. 2015;23:65-73;Pakyari M,et al. Adv Wound Care(New Rochelle)2013;2:215-224). Smad3 binds to Smad mediator (SMAD4) to form a complex that translocates to the nucleus and regulates the expression of genes, including those involved in keratinocyte migration, fibroblast invasion, and extracellular matrix assembly (ibid., Penn JW,et al. Int J Burns Trauma. 2012;2:18-28; citing Hong HJ,et al. Biomaterials. 2008;29:4831-4837). Furthermore, Smad3 can balance re-epithelialization and fibrosis of repaired tissue (ibid., Biernacka A,et al. Growth Factors. 2011;29:196-202; citing Werner S,et al. J Invest Dermatol. 2007;127:998-1008).

[0054] clinical wound healing One of the most important functions of the skin is to be a barrier against the environment (Bakhtyar N,et al.,Stem Cell Res Ther. 2018 Jul 13,9(1):193,citing Bielefeld KA,et al. CMLS. 2013,70:2059-81). Injuries such as burns, chronic skin ulcers as a result of pressure, venous stasis, or diabetes mellitus represent some of the conditions in which tissue integrity is destroyed and wounds are created (ibid.,Bielefeld KA,et al. CMLS. 2013,70:2059-81;citing Singer AJ,Clark RAF. N Engl J Med. 1999,341:738-46). According to the World Health Organization (WHO), burns are a global problem, causing approximately 180,000 deaths per year, and in 2004, approximately 11 million people worldwide suffered burns severe enough to require medical care (ibid., citing WHO (WHO), http: / / www.who.int / en / news-room / fact-sheets / detail / burns,2018). The high mortality rate in burn patients is due to loss of skin, which increases metabolic demands, water loss, and increases the risk of infection. Therefore, wound closure is essential (ibid., citing Sadiq A,et al. Int J Mol Sci. 2018,19). Additionally, approximately 1.5 billion people suffer from poor wound healing due to a combination of progressive aging and lack of adequate medical care (ibid. Sadiq A,et al. Int J Mol Sci. 2018,19;Jeschke MG,et al. EBioMedicine. 2015,2:1536-48;Jeschke MG,et al. Burns:journal of the International Society for Burn Injuries. 2016,42:276-81;Valacchi G,et al. Ann NY Acad Sci. 2012,1259:136-44). For example, diabetes is another widely recognized condition that can lead to serious injuries.Diabetes can cause diabetic ulcers due to prolonged inflammation, lack of angiogenesis, decreased collagen production, high levels of proteinase and collagen synthesis, and macrophage dysfunction (ibid., Fahey TJ 3rd,et al. J Surg Res. 1991,50:308-13; Singer AJ,Clark RA. N Engl J Med. 1999,341:738-46; Shah A,et al. Inflamm Res. 2017 Nov,66(11):931-945; Shah A,Amini-Nik S. International Journal of Drug Research and Technology. 2017;7:8).

[0055] If the steps of wound healing are not coordinated and timely, abnormal wound healing may occur, and open wounds may lead to infection and poor thermal and fluid management. In some pathological disorders, the normal wound healing process is disturbed and prolonged, which may result in chronic non-healing wounds such as diabetic ulcers or pathological scars such as keloid scars (Hu Y,et al. Theranostics. 2018 Jan 1;8(1):169-184,Falanga V. Lancet. 2005;366:1736-43;Plikus MV,et al. Science. 2017;355:748-52 cited). Therefore, shortening healing time and inhibiting scar formation after skin / soft tissue trauma represents an urgent clinical need. Various therapeutic attempts have been made to promote wound healing, but optimal therapeutic strategies are still under development.

[0056] Over the past few years, stem cells have been gaining attention as a powerful tool to improve skin wound healing. Stem cell sources such as human umbilical cord and umbilical cord blood (UCB), amniotic cells, and Wharton's jelly (mucosal connective tissue of the umbilical cord) have shown promising results in wound healing (Hu Y,et al. Theranostics. 2018 Jan 1;8(1):169-184;Bakhtyar N,et al.,Stem Cell Res Ther. 2018 Jul 13,9(1):193;Zhao B,et al. Stem Cells Int. 2018 Jul 25;2018:5420463).

[0057] Mesenchymal Stem Cells (MSCs) Mesenchymal stem cells (MSCs), also known as stromal stem cells, are non-blood adult stem cells found in a variety of tissues. They are characterized by their morphological spindle shape, the expression of specific markers on their cell surface, and the ability to differentiate along at least three lineages (osteogenic, chondrogenic, and adipogenic) under appropriate conditions.

[0058] Due to lack of consensus on MSC phenotype, no single marker has been identified to clearly delineate MSCs in vivo, but it is generally believed that MSCs are positive for cell surface markers CD105, CD166, CD90, and CD44, and MSCs are negative for typical hematopoietic antigens such as CD45, CD34, and CD14. Regarding the differentiation potential of MSCs, studies have reported that a population of bone marrow-derived MSCs has the ability to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic supportive stroma. Studies using transgenic and knockout mice, as well as human musculoskeletal disorders, have reported that MSCs differentiate into multiple lineages during embryonic development and adult homeostasis.

[0059] Analysis of in vitro differentiation of MSCs under appropriate conditions recapitulating in vivo processes has led to the identification of various factors essential for stem cell commitment. Among them, secreted molecules and their receptors (e.g., transforming growth factor-β), extracellular matrix molecules (e.g., collagens and proteoglycans), the actin cytoskeleton, and intracellular transcription factors (e.g., Cbfa1 / Runx2, PPARγ, Sox9, and MEF2) have been shown to play key roles in driving the commitment of multipotent MSCs to specific lineages and maintaining their differentiated phenotype.

[0060] MSCs are known to undergo phenotypic reconstitution during ex vivo manipulation, losing expression of some markers while acquiring new markers (Augello, A. et al, “Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches.” Eur. Cells and Materials (2010) (20): 121-33, citing Jones, et al. 2002 Arthritis Rheum. 46: 3349-60).

[0061] The role of MSCs in wound healing MSCs are thought to orchestrate wound repair by: (1) structural repair through cell differentiation; (2) immunomodulation; (3) secretion of growth factors that promote angiogenesis and re-epithelialization; and (4) recruitment of resident stem cells (Balaji, S. et al, “The role of mesenchymal stem cells in the regenerative wound healing phenotype,” Adv. Wound Care (2012) 1 (40): 159-65).

[0062] Migration of MSCs in vivo Results indicate that MSCs play several simultaneous roles: suppressing inflammation by releasing cytokines; supporting healing by expressing growth factors; modifying the host immune response by secreting immunomodulatory proteins; enhancing the response of endogenous repair cells; and acting as mature functional cells in some tissues such as bone (Phinney,DG and Pittenger,MF. MSC-derived exosomes for cell free therapy. Stem Cells(2017)35:851-58). When labeled and delivered in vivo, MSCs migrate to sites of tissue injury (ibid.). CD44-HA interaction is involved in MSC migratory activity (Zhu,H.et al,“The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix,” Stem Cells(2006)24:928-35).

[0063] Several reports have shown that the SDF-1 / CXCR4 axis is present and functional in MSC populations (Augello, A. et al, “Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches.” Eur. Cells and Materials (2010) (20): 121-33, citing Wynn et al. 2004 Blood 104: 2643-45; Dar et al. 2005 Nat. Immunol. 6: 1038-46). MSCs can also respond to chemotactic signaling molecules acting on pathways other than the SDF-1 / CXCR-4 axis, including monocyte chemoattractant protein-3 (MCP-3) (ibid.).

[0064] It has been proposed that MSCs have the ability to secrete a wide range of bioactive molecules, such as growth factors, cytokines, and chemokines (ibid., Monsel, A. et al, “Mesenchymal stem cell derived secretome and extracellular vesicles for acute lung injury and other inflammatory lung diseases,” Expert Opin. Biol. Ther. (2016) 16:859-71; Caplan, A. and Correa, D., “The MSC: an injury drugstore,” Cell Stem Cell (2011) 9:11-15; Kosuma, GD, et al, “Effect of the microenvironment on mesenchymal stem cells paracrine signaling: opportunities to engineer the therapeutic effect,” Stem Cells Dev. (2017) 26:617-31). These bioactive molecules regulate the local immune response to establish a regenerative microenvironment, which then suppresses inflammation and repairs damaged tissues (ibid.).

[0065] Therapeutic effects of MSCs mediated by stem cell secretion The “paracrine hypothesis,” which posits that the observed therapeutic effects of MSCs are partly mediated by stem cell secretions, has attracted much attention and is supported by experimental data (Arlan, F. et al, “Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress, and activate PI3K / Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia / reperfusion injury,” Stem Cell Res. (2013) 10:301-12, citing Gnecchi et al., “Paracrine mechanisms in adult stem cell signaling and therapy,” Circ. Res. (2008), 103:1204-1219).MSC-CM has been shown to enhance the survival of cardiomyocytes and / or progenitor cells after hypoxia-induced injury (ibid. citing Chimenti et al., “Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice,” Circ. Res. (2010), 106:971-980; Deuse et al., 2009; Gnecchi et al., “Paracrine mechanisms in adult stem cell signaling and therapy,” Circ. Res. (2008), 103:1204-1219; Matsuura et al., “Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice,” J. Clin. Invest., 119 (2009), pp. 2204-2217; Rogers et al., 2011).Furthermore, MSC-CM induces angiogenesis in infarcted myocardium (ibid., citing Chimenti et al., “Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice,” Circ. Res., 106(2010), pp.971-980; Deuse et al., “Hepatocyte growth factor or vascular endothelial growth factor gene transfer maximizes mesenchymal stem cell-based myocardial salvage after acute myocardial infarction,” Circulation, 120(2009), pp.S247-S254; Li et al., “Paracrine factors released by GATA-4 overexpressed mesenchymal stem cells increase angiogenesis and cell survival,” Am. J. Physiol. Heart Circ. Physiol., 299(2010), pp.H1772-H1781). In both mouse and porcine models of myocardial ischemia / reperfusion (I / R) injury, MSC-CMs have been shown to reduce infarct size (ibid., Timmers et al., “Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium,” Stem Cell Res., 1 (2007), pp. 129-137).

[0066] High-performance liquid chromatography (HPLC) and dynamic light scattering (DLS) analyses revealed that MSCs secrete cardioprotective microparticles with hydrodynamic radii in the range of 50-65 nm (ibid., Chen et al., 2011; Lai et al., “Derivation and characterization of human fetal MSCs: an alternative cell source for large-scale production of cardioprotective microparticles,” J. Mol. Cell. Cardiol., 48 (2010), pp. 1215-1224). The therapeutic efficacy of MSC-derived extracellular vesicles (EVs) was independent of the tissue source of MSCs. For example, exosomes derived from human embryonic stem cell-derived MSCs were similar to those derived from other fetal tissue sources (e.g., limb, kidney). This suggested that the secretion of therapeutic EVs may be a general property of all MSCs (Ibid., Lai et al., “Exosome secreted by MSC reduces myocardial ischemia / reperfusion injury,” Stem Cell Res., 4(2010), pp.214-222).

[0067] MSC-derived EVs including exosomes and microvesicles During wound healing, cell-cell communication is important (Bakhtyar N,et al.,Stem Cell Res Ther.2018 Jul 13,9(1):193,Amini-Nik S,et al. J Clin Invest.2014;124:2599-610;citing Raposo G,Stoorvogel W. J Cell Biol.2013;200:373-83). Multicellular organisms rely heavily on cell-cell communication, which can be achieved both through direct cell-cell contact and through the transfer of secreted molecules (ibid.,Raposo G,Stoorvogel W. J Cell Biol.2013;200:373-83;citing Venkat P,et al. Stem Cells Transl Med.2018 Jun;7(6):451-455).

[0068] MSC-derived EVs, including exosomes and microvesicles (MVs), are involved in cell-to-cell communication, cell signaling, and changes in cell or tissue metabolism over short or long distances in the body, affecting tissue responses to injury, infection, and disease (Phinney, DG and Pittenger, MF, “MSC-derived exosomes for cell free therapy,” Stem Cells (2017) 35:851-58). Their contents include cytokines and growth factors, signaling lipids, mRNA, and regulatory miRNA (ibid.). The contents of MSC EVs are not static; they are a product of the MSC tissue origin, its activity, and the interactions between the MSC and its immediate neighbors (ibid.).

[0069] MSCs secrete abundant amounts of biologically active proteins. (Ibid., Tremain N,et al. MicroSAGE analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages. Stem Cells 2001;19:408-418; Phinney DG,et al. “Biological activities encoded by the murine mesenchymal stem cell transcriptome provide a basis for their developmental potential and broad therapeutic efficacy,” Stem Cells 2006;24:186-198; Ren J,et al. “Global transcriptome analysis of human bone marrow stromal cells (BMSC) reveals proliferative,mobile and interactive cells that produce abundant extracellular matrix proteins,some of which may affect BMSC potency,” Cytotherapy 2011;13:661-674).

[0070] Most cells produce EVs as a result of intracellular vesicle localization, including both microvesicles >200 nm and exosomes with diameters of 50–200 nm. Microvesicles are shed from the plasma membrane, whereas exosomes originate from early endosomes and, as they mature into late endosomes / multivesicular bodies, an increasing number of intraluminal vesicles are released as exosomes upon fusion of endosomes with the cell surface (ibid., Lee Y, El Andaloussi S, Wood MJ. “Exosomes and microvesicles: Extracellular vesicles for genetic information transfer and gene therapy,” Hum Mol Genet 2012;21:R15-134; citing Tkach M, Thery C. “Communication by extracellular vesicles:Where we are and where we need to go,” Cell 2016;164:1226-1232).

[0071] Although MSC-derived EVs largely reproduced the vast range of therapeutic effects previously attributed to MSCs, most studies have not rigorously tested this hypothesis (ibid.). For example, myocardial infarction (ibid., citing Bian S, et al. “Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model,” J Mol Med (Berlin) 2014;92:387-397), focal cerebral ischemia (Doeppner TR, et al. “Extracellular vesicles improve post-stroke neuroregeneration and prevent postischemic immunosuppression.” Stem Cells Transl Med 2015;4:1131-1143), gentamicin-induced kidney injury (Reis LA, et al. “Bone marrow-derived mesenchymal stem cells repaired but did not prevent gentamicin-induced acute kidney injury through paracrine effects in rats,” PLoS One 2012;7:e44092), and silicosis (Choi M, et al. “Therapeutic use of stem cell transplantation for cell Various groups have compared the efficacy of MSCs versus MSC-derived EVs, and in some cases MSC-conditioned medium, in animal models of inflammatory bowel disease (inflammatory mediators such as inflammatory bowel disease (IGFs) and / or inflammatory replacement or cytoprotective effect of microvesicle released from mesenchymal stem cell,” Mol Cells 2014;37:133-1394).Most studies have reported that MSC-derived EVs are as effective as MSCs in sparing tissue and / or promoting functional recovery from injury, although this desired outcome is compromised by a lack of adequate controls, comparable dosing, assessment of different disease endpoints, variability in frequency and timing of dosing, and lack of dose-dependent effects, making it difficult to draw reliable conclusions regarding equivalence of efficacy and potency (ibid.).

[0072] amniotic fluid EV Cord blood EVs Human umbilical cord blood (UCB) is an attractive source of transplantable stem cells for wound repair, with several distinct advantages: no risk to the donor, easy access, and low incidence of graft-versus-host disease (Hu Y,et al. Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function. Theranostics. 2018 Jan 1;8(1):169-184,Doi H, et al. Potency of umbilical cord blood- and Wharton's jelly-derived mesenchymal stem cells for scarless wound healing. Sci Rep. 2016;6:18844;He B,et al. Therapeutic potential of umbilical cord blood cells for type 1 diabetes mellitus. J Diabetes. 2015;7:762-73). Studies have reported that local injection of exosomes secreted from human UCB-derived stem cells can promote skin cell proliferation and migration, angiogenesis, and wound closure in animal models of diabetes or burns (ibid., Zhang J,et al. Exosomes Derived from Human Endothelial Progenitor Cells Accelerate Cutaneous Wound Healing by Promoting Angiogenesis Through Erk1 / 2 Signaling. Int J Biol Sci. 2016;12:1472-87; citing Zhang B,et al. HucMSC-Exosome Mediated-Wnt4 Signaling Is Required for Cutaneous Wound Healing. Stem cells. 2015;33:2158-68).Local injection of UCB exosomes into mouse skin wounds accelerated re-epithelialization, reduced scar width, and enhanced new blood vessel formation (ibid.). UCB exosomes also promoted fibroblast proliferation and migration, and enhanced the angiogenic activity of endothelial cells in vitro (ibid.).

[0073] Wharton Jelly EV The umbilical cord has two arteries and one vein and is surrounded by a mucosal connective tissue called Wharton's jelly (WJ) (Bakhtyar N, et al. Exosomes from acellular Wharton's jelly of the human umbilical cord promotes skin wound healing. Stem Cell Res Ther. 2018;9:193, Meyer FA, et al. Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton's jelly. Biochim Biophys Acta. 1983;755:376-387). Wharton's jelly is composed mainly of glycosaminoglycan hyaluronic acid, which forms a gel around fibroblasts and collagen fibrils, protecting the tissue from pressure and maintaining the tissue structure (ibid., Sakamoto T, et al. Electron microscopic histochemical studies on the localization of hyaluronic acid in Wharton's jelly of the human umbilical cord. Nihon Sanka Fujinka Gakkai Zasshi. 1996;48:501-507; Sobolewski K, et al. Collagen and glycosaminoglycans of Wharton's jelly. Biol Neonate. 1997;71:11-21).Many laboratories have identified MSC markers on Wharton's jelly cells and are investigating their properties as both embryonic and adult stem cells (ibid. McElreavey KD,et al. Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord. Biochem Soc Trans. 1991;19,29s;Pirjali T,et al. Isolation and characterisation of human mesenchymal stem cells derived from human umbilical cord Wharton's jelly and amniotic membrane. Int J Organ Transplant Med. 2013;4:111-116;HS Wang,et al Chen,Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord,Stem Cells,22(2004)1330-1337). It has also been reported that WJ-MSC-conditioned medium containing secreted factors has a positive effect on wound healing in vitro (ibid., citing Arno AI,et al. Human Wharton's jelly mesenchymal stem cells promote skin wound healing through paracrine signaling. Stem Cell Res Ther. 2014;5:28). Acellular gelatinous Wharton's jelly (AGWJ) has beneficial wound healing properties in vivo in a mouse model by significantly reducing wound length after AGWJ treatment and enabling wound healing at earlier time points.AGWJ also increased cell migration in vitro and resulted in the expression of α-smooth muscle actin (αSMA), a marker of myofibroblasts (ibid., citing Bakhtyar N,et al. Acellular gelatinous material of human umbilical cord enhances wound healing:a candidate remedy for deficient wound healing. Front Physiol. 2017;8:200). Exosomes isolated from AGWJ enhanced cell viability and migration in vitro and enhanced skin wound healing in a mouse punch biopsy wound model. These exosomes contained large amounts of α-2-macroglobulin (α2M) (ibid.).

[0074] Amniotic epithelial EV Human amniotic epithelial cells (hAECs) are multipotent progenitor cells derived from epiblasts (Zhao et al. Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation. J Mol Histol. 2017 Apr;48(2):121-132). hAECs have been confirmed to play an effective role in promoting wound healing with less scarring (ibid., Zhang B et al.(2015a)HucMSC-exosome mediated-Wnt4 signaling is required for cutaneous wound healing. Stem Cells 33:2158-2168;Zhang J et al.(2015b)Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med 13:49). In vitro studies showed that hAEC exosomes had a smooth spherical structure and were positive for exosome markers CD9, CD63, CD81, Alix, TSG101, and HLA-G (ibid.). Internalization of fluorescently labeled hAEC exosomes by human fibroblasts enhanced their proliferation and migration abilities in a dose-dependent manner (ibid.). Furthermore, deposition of extracellular matrix (ECM), especially collagen-I and III, was downregulated by treatment with high concentrations of hAEC exosomes and stimulating the expression of MMP-1. In vivo wound assays also showed that local injection of hAEC exosomes into rat skin wounds promoted the wound healing process with well-arranged collagen fibers.

[0075] Not all MSC-derived EVs are equivalent. For example, it has been reported that exosomes isolated from adipose-derived MSCs contain up to four-fold higher levels of enzymatically active neprilysin compared to bone marrow-derived MSCs (ibid., citing Katsuda T, et al. “Human adipose tissue-derived mesenchymal stem cells secrete functional neprilysin-bound exosomes,” Sci Rep(2013)3:1197). EVs derived from bone marrow and umbilical cord-derived MSCs were shown to inhibit proliferation and induce apoptosis of U87MG glioblastoma cells in vitro, whereas EVs derived from adipose-derived MSCs promoted cell proliferation but did not affect U87MG viability (ibid., Del Fattore, A. et al., “Differential effects of extracellular vesicles secreted by mesenchymal stem cells from different sources on glioblastoma cells,” Expert Opin. Biol. Ther. (2015) 15:495-504). Furthermore, exosomes prepared from different tissue-specific MSCs have been shown to have measurably different effects on neurite outgrowth in primary cortical neuron and dorsal root ganglion explant cultures (ibid., citing Lopez-Verrilli et al. “Mesenchymal stem cell-derived exosomes from different sources selectively promote neuritic outgrowth,” Neuroscience 2016;320:129-139).

[0076] amniotic fluid EV Human amniotic fluid-derived stem cells (hAFS) have been extensively characterized as multipotent mesenchymal progenitor cells that express pluripotency markers and high self-renewal capacity similar to embryonic stem cells, without tumorigenicity or ethical concerns (Balbi C,et al. First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential. Stem Cells Transl Med. 2017 May;6(5):1340-1355, cited De Coppi P,et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100-106). hAFS are fetal but not embryonic, thus overcoming many ethical concerns and can be easily obtained from residual or discarded amniotic fluid samples collected during amniocentesis or qualified cesarean section, when expressing the stem cell marker c‐KIT (ibid., De Coppi P,et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100-106; Pozzobon M,et al. Isolation of c‐Kit + human amniotic fluid stem cells from second trimester. Methods Mol Biol 2013;1035:191-198; Schiavo AA, et al. Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia. Stem Cell Res Ther 2015;6:209). c-KIT +hAFS has been shown to exert a cardioprotective paracrine effect reducing infarct size in an acute rat model of myocardial infarction (MI) (ibid., citing Bollini S, et al. Amniotic fluid stem cells are cardioprotective following acute myocardial infarction. Stem Cells Dev 2011;20:1985-1994). hAFS actively secrete EVs with sizes ranging from 50 to 1,000 nm (ibid.). These include nano-sized exosome particles identified by the expression of TSG101, ALIX, CD81, CD9, AnnV, and CD63, as well as cell-specific markers such as CD105 (ibid., citing Lotvall J,et al. Minimal experimental requirements for definition of extracellular vesicles and their functions:A position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 2014;3:26913;Connolly KD,et al. Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis. J Extracell Vesicles 2015;24;4:29159).

[0077] The subject of the present disclosure is to provide an EV composition for improving wound healing, and a method for preparing the same. Summary of the Invention

[0078] According to one aspect, the invention described provides a method of promoting wound healing in a subject in need thereof, comprising contacting wound tissue of the subject with a first composition comprising a therapeutic amount of extracellular vesicles (EVs) derived from human amniotic fluid (AF), the therapeutic amount being effective to reduce wound area and promote repair of the wound tissue. According to one embodiment, the composition is effective to promote wound healing by activating epithelial cells to transition to a mesenchymal cell phenotype (EMT). According to another embodiment, the EVs are derived from amniotic fluid mesenchymal stem cells (MSCs). According to another embodiment, the EVs are characterized by a sedimentation at about 100,000×g, a buoyant density in sucrose of about 1.10-1.21 g / ml, and an average diameter of about 50 nm to about 200 nm. According to another embodiment, the contacting is topical or subcutaneous. According to another embodiment, the first composition is effective to increase mRNA levels of one or more of vimentin, N-cadherin, Col1a1, Acta2, or TGFbr2. According to another embodiment, the method further comprises contacting the wound tissue of the subject with a second composition comprising a therapeutic amount of EV-depleted AF, the therapeutic amount of the second composition being effective to activate mesenchymal-epithelial transition (MET) and promote repair of the injured tissue. According to another embodiment, the length of time between contacting the tissue with the first composition and the second composition is about 4 hours to about 24 hours. According to another embodiment, the second composition is effective to increase Stat3 mRNA levels. According to another embodiment, the wound is a chronic wound. According to another embodiment, the wound is a diabetic ulcer, a pressure ulcer, or a venous ulcer. According to another embodiment, the wound is a burn. According to another embodiment, the composition further comprises a pharma- ceutically acceptable carrier. According to another embodiment, the EVs are purified from the amniotic fluid by one or more of a) ultracentrifugation; b) sucrose density gradient centrifugation; c) column chromatography; d) size exclusion; or e) filtration through a device comprising an affinity matrix selective for EVs. According to another embodiment, the method further comprises filtering the EVs by size. According to another embodiment, the EVs are characterized by an average diameter of about 50 nm to about 200 nm.

[0079] According to another aspect, the invention described provides a two-step method of promoting wound healing in a subject in need thereof, comprising, in order, contacting the wound with a composition comprising extracellular vesicles (EVs) derived from amniotic fluid (AF) to promote early stage wound healing in the subject; and contacting the wound with a composition comprising EV-depleted AF to promote late stage wound healing in the subject. According to one embodiment, early stage wound healing is characterized by activating epithelial-mesenchymal transition (EMT) and inducing cell migration, and late stage wound healing is characterized by activating mesenchymal-epithelial transition (MET) and epithelialization of the wound. According to another embodiment, the EVs are derived from amniotic fluid mesenchymal stem cells (MSCs). According to another embodiment, the EVs are characterized by a sedimentation at about 100,000×g, a buoyant density in sucrose of about 1.10-1.21 g / ml, and an average diameter of about 50 nm to about 200 nm. According to another embodiment, the contacting is topical or subcutaneous.

[0080] According to another aspect, the invention described provides a method of regulating skin condition in a subject in need thereof, comprising contacting the skin of the subject with a composition comprising a therapeutic amount of extracellular vesicles (EVs) derived from human amniotic fluid (AF), the therapeutic amount being effective to improve skin texture, reduce wrinkles, or both, thereby regulating skin condition. According to one embodiment, the method further comprises microneedling the skin prior to contacting with the composition. According to another embodiment, the composition is effective to regulate skin condition by activating epithelial-mesenchymal transition (EMT). [Brief description of the drawings]

[0081] [Figure 1]A representative series of images showing brightfield microscopy images (20x objective) of C2C12 myoblasts during a scratch test wound-healing assay at 0, 12, 18, and 24 h, incubated with unconditioned serum-free medium + 10% amniotic fluid (uncSFM+AF), uncSFM containing the same amount of amniotic fluid-derived exosomes as in uncSFM+10%AF (uncSFM+AFexos), or uncSFM + 10% exosome-depleted amniotic fluid (uncSFM+exo(-)AF). Dotted lines outline areas not occupied by cells; scale bar indicates 50 μm. [Diagram 2] Graph showing quantification of scratch area (percentage of total scratch area at time zero) for the conditions described in Figure 1. Area was calculated using ImageJ software and six independent replicates were measured per condition and time point. Each data point represents the mean percentage of area relative to the area at time zero ± standard deviation (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student's t-test vs. uncSFM+AF or uncSFM+AFexos). [Diagram 3] Western blot analysis of whole amniotic fluid (AF; total), crude fractions from amniotic fluid-derived exosome purification using the ExoQuick TC-ULTRA kit (SBI Biosciences; exoCrude), purified fractions / eluates from the ExoQuick TC-ULTRA kit (SBI Biosciences; exoPure), or exosome-depleted AF (exo(-)AF). Antibodies used to probe for CD63, albumin, and CD9 are shown on the left and molecular weights in kilodaltons (kDa) are shown on the right. [Figure 4] FIG. 1 is a graph showing the relative abundance of vimentin mRNA in conditioned medium from amniotic fluid fractions in a myoblast scratch test assay. [Diagram 5] FIG. 13 is a graph showing the relative abundance of N-Cad / E-Cad mRNA ratio in conditioned medium from amniotic fluid fractions in a myoblast scratch test assay. [Figure 6]FIG. 13 is a graph showing the relative abundance of Col1a1 mRNA in conditioned medium from amniotic fluid fractions in a myoblast scratch test assay. [Figure 7] FIG. 13 is a graph showing the relative abundance of Acta2 mRNA in conditioned medium from amniotic fluid fractions in a myoblast scratch test assay. [Figure 8] FIG. 13 is a graph showing the relative abundance of Tgfbr2 mRNA in conditioned medium from amniotic fluid fractions in a myoblast scratch test assay. [Figure 9] FIG. 1 is a graph showing the relative abundance of Stat3 mRNA in conditioned medium from amniotic fluid fractions in a myoblast scratch test assay. [Figure 10] A representative series of images showing brightfield microscopy images (20x objective) of MMM fibroblasts during the scratch test wound-healing assay at 0, 12, 18, and 24 h, incubated with unconditioned serum-free medium + 10% amniotic fluid (uncSFM+AF), uncSFM containing the same amount of amniotic fluid-derived exosomes as in uncSFM+10% AF (uncSFM+AFexos), or uncSFM + 10% exosome-depleted amniotic fluid (uncSFM+exo(-)AF). Dotted lines outline areas not occupied by cells; scale bar indicates 50 μm. [Figure 11] Graph showing quantification of scratch area (area percentage of total scratch at time zero) for the conditions described in Figure 10. Area was calculated using ImageJ software and six independent replicates were measured per condition and time point. Each data point represents the mean area percentage relative to the area at time zero ± standard deviation (*P<0.05 and **P<0.01 by Student's t-test for uncSFM+AF or uncSFM+AFexos). [Figure 12] Photographs showing subjects before and 14 days after treatment with Celexoderm™. [Figure 13]Venn diagram showing the degree of overlap (or non-overlap) of biological triplicate samples analyzed by liquid chromatography coupled to tandem mass spectrometry (LC / MS-MS) of total amniotic fluid (total AF), exosome-depleted AF (exo(-)AF), and exosome-enriched AF fraction (AF exos). Analysis was performed to generate peptide enrichments for the total spectrum and then measure the degree of overlap of peptides that uniquely mapped to proteins. The resulting Venn diagram shows the degree of overlap (or non-overlap) of the analyzed samples. [Figure 14] FIG. 1 shows the results of gene ontology analysis using the DAVID bioinformatics database to determine biological values, functions, and processes significantly associated with proteins identified by LC / MS-MS to be present in the exosome-enriched AF fraction at higher levels than proteins found in total AF. [Figure 15] FIG. 1 shows the results of gene ontology analysis using the DAVID bioinformatics database to determine biological value, function, and processes significantly associated with proteins identified by LC / MS-MS to be present in the exosome-depleted AF fraction at higher levels than proteins found in total AF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. Thus, for example, reference to a "peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0083] As used herein, the term "about" refers to ±20% of the numerical value of the number with which it is used. Thus, about 50% means a range of 40% to 60%.

[0084] "Administering" when used in conjunction with therapeutic procedures means giving or applying a therapeutic agent directly to a target organ, tissue or cell, or administering a therapeutic agent to a subject, whereby the therapeutic agent positively affects the organ, tissue, cell, or subject to which it is targeted. Thus, as used herein, the term "administering" when used in conjunction with EVs or compositions thereof can include, but is not limited to, providing EVs in or on a target organ, tissue, or cell; or providing EVs systemically to a patient, for example, by intravenous injection, whereby the therapeutic agent reaches the target organ, tissue, or cell. "Administering" can be accomplished by parenteral, oral, subcutaneous, or topical administration, by inhalation, or by such methods in combination with other known techniques.

[0085] As used herein, the term "allogeneic" refers to belonging to or derived from the same species, but genetically distinct.

[0086] The term "amino acid" is used to refer to an organic molecule that contains both an amino group and a carboxyl group; the amino acids that function as the building blocks of natural proteins are alpha-amino acids, in which both the amino group and the carboxyl group are attached to the same carbon atom. The terms "amino acid residue" or "residue" are used interchangeably to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide, and include, but are not limited to, naturally occurring amino acids and analogs of naturally occurring amino acids that can function in a manner similar to the naturally occurring amino acids.

[0087] The abbreviations used herein for amino acids are those conventionally used: A=Ala=alanine; R=Arg=arginine; N=Asn=asparagine; D=Asp=aspartic acid; C=Cys=cysteine; Q=G1n=glutamine; E=Glu=glutamic acid; G=Gly=glycine; H=His=histidine; I=Ile=isoleucine; L=Leu=leucine; K=Lys=lysine; M=Met=methionine; F=Phe=phenylalanine; P=Pro=proline; S=Ser=serine; T=Thr=threonine; W=Trp=tryptophan; Y=Tyr=tyrosine; V=Val=valine. The amino acids may be L- or D-amino acids. The amino acids may be replaced with synthetic amino acids that are modified to increase the half-life of the peptide, increase the potency of the peptide, or increase the bioavailability of the peptide.

[0088] The following represent groups of amino acids that are conservative substitutions for one another: Alanine (A), serine (S), threonine (T); Aspartic acid (D), glutamic acid (E); Asparagine (N), Glutamine (Q); Arginine (R), Lysine (K); Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0089] Amnion. The amnion develops from extraembryonic tissues and consists of a fetal component (the chorionic plate) and a maternal component (the decidua, meaning the inner lining of the uterus during pregnancy), held together by the chorionic villi, which connect the cytotrophoblastic outer layer of the chorion to the decidua basalis. The fetal components, including the amnion and chorion, separate the fetus from the endometrium. The amnion forms the outer border of the sac that surrounds the fetus, while the innermost layer of the sac is the amnion.

[0090] The amniotic membrane (AM) consists of, from inside to outside, (A) an epithelial monolayer, (B) a thick basement membrane, (C) stratum compactum, (D) fibroblast layer, and (E) stratum spongiosum. The amniotic epithelium is the innermost layer closest to the fetus, in contact with the amniotic fluid, and consists of a monolayer of cells arranged uniformly on the basement membrane. The epithelial layer can be removed while keeping the basement membrane and the interstitial surface morphologically intact. The basement membrane consists of a network of reticular fibers. The stratum compactum of the interstitial matrix adjacent to the basement membrane forms the main fibrous skeleton of the AM. Collagen of the stratum compactum is secreted by mesenchymal cells within the fibroblast layer. Interstitial collagens (types I and III) predominate and form parallel bundles that maintain the mechanical integrity of the AM. Collagens of types V and VI form thread-like connections between the interstitial collagens and the epithelial basement membrane. The fibroblast layer consists of a loose fibroblast network embedded in a mass of reticular tissue. The spongy layer of the interstitial matrix represents the tissue of the extraembryonic cavity, located adjacent to the chorion and compressed between the amnion and chorion. It mainly contains a non-fibrillar meshwork of type III collagen. The spongy layer is loosely connected to the chorion; therefore, the AM is easily separated from the chorion by blunt dissection (Niknejad, H. et al, Eur. Cells and Materials (2008) 15:88-99).

[0091] Throughout this specification and claims, the term "amniotic (AM) cells" is used interchangeably with the term "amniotic epithelial cells (AECs)" and is intended to include all cell types derived from the amnion, the majority of which are amniotic epithelial cells.

[0092] As used herein, the term "amniotic stem cells" refers to pluripotent stem cells, multipotent stem cells, and progenitor cells derived from the amnion, which can give rise to a limited number of cell types in vitro and / or in vivo under appropriate conditions, and explicitly includes both amniotic epithelial cells and amniotic stem cells.

[0093] As used herein, the terms "animal," "patient," and "subject" include, but are not limited to, humans and non-human vertebrates, such as wild, domestic, and farm animals. According to some embodiments, the terms "animal," "patient," and "subject" may refer to humans. According to some embodiments, the terms "animal," "patient," and "subject" may refer to non-human mammals.

[0094] As used herein, the phrase "subject in need" of treatment for a particular condition is a subject who has, has been diagnosed with, or is at risk of developing that condition. According to some embodiments, the phrase "subject in need" of such treatment is also used to refer to a patient who (i) is being administered a described composition of the invention; (ii) is receiving a described composition of the invention; or (iii) has received at least one described composition of the invention, unless the context and usage of the phrase dictates otherwise.

[0095] The term "antibody" as used herein refers to a polypeptide or group of polypeptides consisting of at least one binding domain formed from the folding of polypeptide chains with a three-dimensional binding space with an internal surface shape and charge distribution complementary to the antigenic determinant characteristics of an antigen. Antibodies usually have a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. As used herein, a "targeted binding agent" is an antibody or binding fragment thereof that preferentially binds to a target site. According to some embodiments, the targeted binding agent is specific for only one target site. According to some embodiments, the targeted binding agent is specific for multiple target sites. According to some embodiments, the targeted binding agent may be a monoclonal antibody and the target site may be an epitope. As used herein, the term "epitope" refers to a portion of an antigen or other macromolecule that can form a binding interaction that interacts with the variable region binding pocket of an antibody. "Binding fragments" of antibodies are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single chain antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counter-receptor if an excess of the antibody reduces the amount of receptor bound to the counter-receptor by at least about 20%, 40%, 60% or 80%, more usually by about 85% or more (measured in an in vitro competitive binding assay). The antibody may be an oligoclonal antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multispecific antibody, a bispecific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody, and an antibody that can be labeled in soluble or bound form, alone or in combination with other amino acid sequences provided by known techniques, as well as fragments, variants, or derivatives thereof. The antibody may be from any species.The term antibody also includes binding fragments of the antibodies of the invention; exemplary fragments include Fv, Fab, Fab', single chain antibodies (svFC), dimeric variable regions (diabodies) and disulfide stabilized variable regions (dsFv). As discussed herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated to be encompassed by the described invention so long as the variation in the amino acid sequence maintains at least about 75%, and in some embodiments at least about 80%, about 90%, about 95%, and about 99% sequence identity to the antibody or immunoglobulin molecule described herein. Conservative amino acid substitutions are contemplated. For example, it is reasonable to expect that independent substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not have a major effect on the binding function or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can be easily determined by assaying the specific activity of the polypeptide derivative. The assay is described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be easily prepared by those skilled in the art. According to some embodiments, the amino and carboxy termini of the fragment or analog occur near the boundaries of the functional domain. Structural and functional domains can be identified by comparing the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. For example, computer comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. See, for example, Bowie et al. Science 253:164 (1991), which is incorporated by reference in its entirety.

[0096] As used herein, the term "antigen" refers to a molecule, e.g., a peptide, polypeptide, protein, fragment, or other biological moiety, that elicits an antibody response in a subject or that is recognized and bound by an antibody.

[0097] The term "apoptosis" or "programmed cell death" refers to a highly regulated, active process that contributes to biological homeostasis, consisting of a series of biochemical events that result in a variety of morphological changes, including blebbing, alterations in the cell membrane, e.g., loss of membrane asymmetry and adhesion, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, without causing damage to the organism.

[0098] As used herein, the term "autocrine signaling" refers to a type of cell signaling in which a cell secretes signaling molecules that act on itself or other neighboring cells of the same type.

[0099] The terms "autologous" or "self" are used interchangeably herein to mean derived from the same organism.

[0100] The term "basal medium" or "serum-free medium (SFM)" is intended to mean a medium that does not contain added serum (i.e., is essentially serum-free). Examples of basal media include, but are not limited to, DMEM / F12, DMEM, F12, and IMDM.

[0101] The term "binding" and other grammatical forms as used herein refer to a permanent attraction between chemical entities. Binding specificity includes both binding to a specific partner and non-binding to other molecules. Functionally important binding can occur with affinities ranging from low to high, and design elements can suppress undesirable cross-type interactions. Post-translational modifications can also change the chemical nature and structure of the interaction. "Promiscuous binding" can involve a degree of structural plasticity, resulting in different subsets of residues important for binding to different partners. "Relative binding specificity" is a feature in biochemical systems that a molecule interacts specifically with its target or partner, thereby affecting them characteristically depending on what the individual target or partner is.

[0102] The term "biomarker" or "marker" as used herein refers to a peptide, protein, nucleic acid, antibody, gene, metabolite, or any other substance used as an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as a cellular or molecular indicator of normal biological processes, pathogenic processes, or pharmacological response to a therapeutic intervention. The term "indicator" as used herein refers to any substance, number, or ratio derived from a set of observed facts that may reveal relative changes as a function of time; or a visible signal, label, mark, annotation, or symptom, or evidence of its presence or presence itself. Once a proposed biomarker is validated, it can be used to diagnose disease risk, presence of disease in an individual, or tailor treatment of disease in an individual (selection of drug treatment or dosing regimen). In evaluating potential drug therapies, biomarkers can be used as surrogates for natural endpoints such as survival rate or irreversible morbidity. If a treatment causes a change in a biomarker and the change is directly related to improved health, the biomarker can serve as a surrogate endpoint to evaluate clinical utility. Clinical endpoints are variables that can be used to measure how a patient feels, functions, or survives. Surrogate endpoints are biomarkers that are intended to be substitutes for clinical endpoints; these biomarkers have been shown to predict clinical endpoints with a confidence level acceptable to regulatory and clinical communities.

[0103] The term "carrier" as used herein refers to a substance that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the compounds of the compositions of the present invention described. The carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the mammal to be treated. The carrier may be inert or may have pharmaceutical utility. The terms "excipient", "carrier", or "vehicle" are used interchangeably and refer to carrier substances suitable for the formulation and administration of the pharma-ceutically acceptable compositions described herein. Carriers and vehicles useful herein include such substances known in the art that are non-toxic and do not interact with other components.

[0104] The term "chorion" as used herein refers to the outer egg membrane that surrounds the amnion, the embryo, and other membranes and entities in the uterus. A spongy layer of loosely arranged collagen fibers separates the amniotic and chorionic mesoderm. The chorion consists of regions of mesoderm and trophoblast. The chorion and amniotic mesoderm are similar in composition. A large, incomplete basement membrane separates the chorionic mesoderm from the extravillous trophoblast cells. The latter, like the trophoblast cells present in the substrate, are dispersed within the fibrinoid layer and express immunohistochemical markers of proliferation. The Langhans fibrinoid layer normally increases during pregnancy and consists of two different types of fibrinoid: an inner matrix type (more compact) and an outer fibrin type (more reticular). At the edge and base of the placenta, the trophoblast is extensively interdigitated with the decidua (Cunningham, F. et al., The placenta and fetal membranes, Williams Obstetrics, 20th ed. Appleton and Lange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of the human placenta. New York, Springer-Verlag, 2000, 42-46, 116, 281-297). The chorion, which connects the maternal tissues, consists of four layers. These, from inside to outside, are: (F) the cellular layer, a thin layer of interlaced fibroblastic network, often incomplete or absent altogether; (G) the reticular layer, consisting of a reticular network whose fibers tend to be parallel, with some fibroblasts and many Hofbauer cells; (H) the pseudobasement membrane, a layer of dense connective tissue firmly attached to the reticular layer above, which feeds anchoring and branching fibers into the trophoblast; and (I) the trophoblast layer, the innermost layer of the chorion, consisting of 2-10 layers of trophoblast cells that are in contact with the maternal decidua on its deeper aspect. This layer contains the chorionic villi (Bourne, GL, Am. J. Obstet. & Gynec. (1960) 79(6):1070-73).

[0105] "Cluster of Differentiation" or "cluster of designation" (CD) molecules are utilized for cell sorting using a variety of methods, including flow cytometry. Cell populations are usually defined using a "+" or "-" sign, which indicates whether a particular cell fraction expresses a particular CD molecule.

[0106] As used herein, the term "conditioned medium" (or media in the plural) refers to spent medium harvested from cultured cells that contains metabolic products, growth factors, RNA and proteins released into the medium by the cultured cells.

[0107] As used herein, the term "contact" and its various grammatical forms refer to the state or condition of contact, or of immediate or nearby proximity.

[0108] As used herein, the term "culture medium" (or media in the plural) refers to a nutrient-containing substance in which cells or tissues are cultured under controlled growth.

[0109] The term "cytokine" as used herein refers to small soluble protein substances secreted by cells that exert various effects on other cells. Cytokines mediate many important physiological functions, including proliferation, development, wound healing, and immune response. They act by binding to cell-specific receptors on the cell membrane, which initiate distinct signaling cascades within the cell that ultimately result in biochemical and phenotypic changes in the target cell. Generally, cytokines act locally. They include type I cytokines, which include many of the interleukins, and several hematopoietic growth factors; type II cytokines, including interferons and interleukin-10; tumor necrosis factor (TNF)-related molecules, including TNFα and lymphotoxin; members of the immunoglobulin superfamily, including interleukin-1 (IL-1); and chemokines, a family of molecules that play important roles in various immune and inflammatory functions. The same cytokine may affect cells differently depending on the state of the cell. Cytokines often regulate the expression of other cytokines, triggering their cascades.

[0110] As used herein, the term "derived from" is meant to encompass any method for receiving, obtaining, or modifying something from a source.

[0111] As used herein, the terms "detection," "determining," and other grammatical forms thereof are used to refer to a method performed to identify or quantify a biomarker, such as, for example, the presence or level of miRNA, or the presence or absence of a condition in a biological sample. The amount of biomarker expression or activity detected in a sample may be none or below the detection level of the assay or method.

[0112] As used herein, the term "differentiation" refers to a developmental process involving an increased level of organization or complexity of cells or tissues with more specialized functions.

[0113] As used herein, the term "disease" or "disorder" refers to an impaired health or state of abnormal function.

[0114] As used herein, the phrase "early stage" refers to early aspects of wound healing such as coagulation / hemostasis and inflammation. As used herein, the phrase "late stage" refers to later aspects of wound healing such as proliferation and remodeling / maturation.

[0115] As used herein, the term "endogenous" refers to naturally occurring, incorporated within, contained within, attached to, attached to, or present within. As used herein, the term "exogenous" refers to non-native, or originating or produced outside of a particular EV, cell, organism, or species.

[0116] As used herein, the term "enrich" refers to increasing the proportion of a desired material, for example, increasing the relative frequency of a subtype of cells or cell components in a cell population compared to their natural frequency. Positive selection, negative selection, or both are generally considered necessary for any enrichment scheme. Selection methods include, but are not limited to, magnetic separation and fluorescence activated cell sorting (FACS).

[0117] As used herein, the phrase "epithelial-mesenchymal transition" or "EMT" refers to the process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. As used herein, the phrase "mesenchymal-epithelial transition" or "MET" refers to the reverse process.

[0118] As used herein, the term "worsening" refers to an increase in the severity of a disease or any of its signs or symptoms.

[0119] As used herein, the term "growth" and its various grammatical forms refer to the process by which dispersed living cells multiply in vitro in a culture medium, resulting in an increase in the number or amount of living cells.

[0120] As used herein, the term "expression" and its various grammatical forms refer to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polynucleotide, or protein. The transcript and the encoded polypeptide may be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may also refer to post-translational modification of a polypeptide or protein.

[0121] As used herein, the term "extracellular vesicles" or "EVs" includes exosomes and microvesicles that carry bioactive molecules such as proteins, RNA and microRNAs that can be released and impact the extracellular environment. Microvesicles are small membrane-enclosed sacs that are believed to be generated by the outward budding and division of membrane vesicles from the cell surface. Exosomes are primarily derived from preformed multivesicular bodies that are released upon fusion with the plasma membrane. The term "EV-depleted" means essentially free or substantially free of extracellular vesicles.

[0122] As used herein, the term "growth factor" refers to an extracellular polypeptide molecule that binds to a cell surface receptor and triggers an intracellular signaling pathway that results in proliferation, differentiation, or other cellular response. These pathways stimulate the accumulation of proteins and other macromolecules, for example, by increasing their rate of synthesis, decreasing their rate of degradation, or both. Exemplary growth factors include, but are not limited to, the following:

[0123] Fibroblast Growth Factors (FGFs). The fibroblast growth factor (FGF) family currently has more than 12 structurally related members. FGF1 is also known as acidic FGF; FGF2 is sometimes called basic FGF (bFGF); and FGF7 is sometimes called keratinocyte growth factor. In vertebrates, more than 12 distinct FGF genes are known; they can generate hundreds of protein isoforms by RNA splicing or by altering start codons in different tissues. FGFs can activate a series of receptor tyrosine kinases called fibroblast growth factor receptors (FGFRs). Receptor tyrosine kinases are proteins that extend through the cell membrane. The portion of the protein that binds to paracrine factors is on the outside of the cell, while the dormant tyrosine kinases (i.e., proteins that can phosphorylate other proteins by splitting ATP) are on the inside of the cell. When an FGF receptor binds to an FGF (and only when it binds to an FGF), a dormant kinase is activated, which phosphorylates specific proteins in the responding cell, activating those proteins.

[0124] FGFs are associated with several developmental functions, including angiogenesis (blood vessel formation), mesoderm formation, and axonal outgrowth. Although FGFs are often substitutable for one another, their expression patterns confer distinct functions to them. For example, FGF2 is particularly important for angiogenesis, whereas FGF8 is involved in midbrain and limb development.

[0125] Insulin-like growth factor 1 (IGF-1). IGF-1, a hormone similar in molecular structure to insulin, exerts growth-promoting effects on nearly all cells in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cells, and lungs. It plays a key role in growth in children and continues to exert anabolic effects in adults. IGF-1 is produced primarily as an endocrine hormone by the liver and in a paracrine / autocrine manner in target tissues. Production is stimulated by growth hormone (GH) and can be delayed by nutritional deficiency, growth hormone insensitivity, lack of growth hormone receptor, or impairment of downstream signaling molecules, including tyrosine protein phosphatase nonreceptor type 11 (also known as SHP2 and encoded by the PTPN11 gene in humans) and signal transducer and activator of transcription 5B (STAT5B), a member of the STAT family of transcription factors. Its primary action is mediated by binding to its specific receptor, the insulin-like growth factor 1 receptor (IGF1R), which is present on many cell types in many tissues. Upon binding to the receptor tyrosine kinase IGF1R, intracellular signaling is initiated; IGF-1 is one of the most potent natural activators of the AKT signaling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death. IGF-1 is the primary mediator of the effects of growth hormone (GH), which is made in the pituitary gland and released into the bloodstream, where it then stimulates the liver to produce IGF-1. IGF-1 then stimulates body growth throughout the body. In addition to its insulin-like effects, IGF-1 can also regulate cell growth and development, especially in neuronal cells, as well as cellular DNA synthesis.

[0126] IGF-1 has been shown to increase the expression levels of the chemokine receptor CXCR4 (the receptor for stromal cell-derived factor-1, SDF-1), significantly increasing the migratory response of MSCs to SDF-1 (Li,Y,et al,“Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells,” 2007 Biochem. Biophys. Res.Communic.356(3):780-784). The IGF-1-induced increase in MSC migration in response to SDF-1 was attenuated by PI3 kinase inhibitors (LY294002 and wortmannin), but not by the mitogen-activated protein / ERK kinase inhibitor PD98059. Without being limited to any particular theory, the data indicate that IGF-1 increases the MSC migratory response via CXCR4 chemokine receptor signaling, which is PI3 / Akt-dependent.

[0127] Transforming growth factor beta (TGF-β). The TGF-β superfamily has over 30 structurally related members that regulate some of the most important interactions in development. Proteins encoded by TGF-β superfamily genes are processed such that the carboxy-terminal region contains mature peptides. These peptides are dimerized into homodimers (with themselves) or heterodimers (with other TGF-β peptides) and secreted from cells. The TGF-β superfamily includes the TGF-β family, the activin family, bone morphogenetic proteins (BMPs), the Vg-1 family, and other proteins including glial-derived neurotrophic factor (GDNF, required for differentiation of kidney and enteric neurons) and Müllerian inhibitory factor, involved in sex determination in mammals. TGF-β family members TGF-β1, 2, 3, and 5 are important in regulating extracellular matrix formation between cells and in regulating cell division (both positively and negatively). TGF-β1 increases the amount of extracellular matrix epithelial cells by both stimulating collagen and fibronectin synthesis and inhibiting matrix degradation. TGF-β may be important in controlling where and when epithelium branches to form ducts in the kidney, lung, and salivary glands.

[0128] Vascular endothelial growth factor (VEGF). VEGF is a growth factor that mediates many functions of endothelial cells, including proliferation, migration, invasion, survival, and permeability. VEGF and their corresponding receptors are key regulators in the cascade of molecular and cellular events that ultimately lead to the development of the vascular system, either by vasculogenesis, angiogenesis, or the formation of the lymphatic vasculature. VEGF is a key regulator in physiological angiogenesis and also plays an important role in skeletal growth and repair.

[0129] Normal function of VEGF allows new blood vessels to be created during embryonic development, after injury, and to bypass blocked blood vessels. In a mature, established vasculature, the endothelium plays a key role in maintaining the homeostasis of surrounding tissues by providing a communication network to adjacent tissues and responding to demands as needed. In addition, the vasculature provides growth factors, hormones, cytokines, chemokines, metabolites, etc. required by surrounding tissues and acts as a barrier to limit the movement of molecules and cells.

[0130] The term "hybridization" as used herein refers to the mutual binding of two single-stranded nucleic acid molecules through base pairing. Two completely complementary strands can easily bind (i.e., "anneal") to each other because nucleotides bind to their complements under normal conditions. However, because the molecular structures of nucleotides are different, a single mismatch between the two strands makes the binding between them more energetically unfavorable. By quantifying the rate at which two strands anneal, the effect of base incompatibility can be measured, providing information on the similarity of the base sequences between the two annealing strands.

[0131] The term "inflammation" as used herein refers to the physiological process by which angiogenic tissues respond to injury. See, for example, FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, which is incorporated herein by reference. During the inflammatory process, cells involved in detoxification and repair are recruited to the site of injury by inflammatory mediators. Inflammation is often characterized by intense infiltration of leukocytes, especially neutrophils (polymorphonuclear cells), at the site of inflammation. These cells promote tissue damage by releasing toxic substances into the vessel wall or into intact tissue. Traditionally, inflammation has been divided into acute and chronic responses. The term "acute inflammation" as used herein refers to a rapid, temporary (minutes to days) and relatively uniform response to acute injury characterized by the accumulation of fluid, plasma proteins, and neutrophil leukocytes. Examples of harmful substances that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), foreign bodies of exogenous (e.g., asbestos) or endogenous (e.g., uric acid crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., corrosive) agents. As used herein, the term "chronic inflammation" refers to inflammation that is of longer duration and has a vague and unclear end. Chronic inflammation takes over when acute inflammation persists, either through incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring at the same location. Chronic inflammation, which includes the influx of lymphocytes and macrophages and proliferation of fibroblasts, can cause tissue scarring at sites of prolonged or repeated inflammatory activity.

[0132] As used herein, the term "infusion" and other grammatical forms refer to the introduction of a fluid other than blood into a vein.

[0133] The term "isolated" is used herein to refer to a material, such as, but not limited to, a nucleic acid, peptide, polypeptide, or protein, that is (1) substantially or essentially free of components that normally accompany or interact with it as found in its natural environment. The terms "substantially free" or "essentially free" are used herein to refer to a substantial or significant absence, or greater than about 95%, 96%, 97%, 98%, 99%, or 100% absence. An isolated material optionally includes materials that are not found with it in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) modified by deliberate human intervention in the composition and / or is located in a subcellular location (e.g., genome or subcellular organelle) that is not natural to the material found in its environment. Modifications to obtain a synthetic material may be performed on the material in its natural state or removed from its natural state.

[0134] As used herein, the term "matrix metalloproteinase" refers to a collection of zinc-dependent proteases involved in the degradation and remodeling of extracellular matrix components (Guiot, J. et al, "Blood biomarkers in idiopathic pulmonary fibrosis," Lung (2017) 195(3): 273-280, citing Oikonomidi et al., "Matrix metalloproteinases in respiratory diseases: from pathogenesis to potential clinical implications," Curr Med Chem. 2009; 16(10): 1214-1228). MMP-1 and MMP-7 appear to be overexpressed primarily in the plasma of IPF patients compared to hypersensitivity pneumonitis, sarcoidosis, and COPD, and may be useful in differential diagnosis (ibid., citing Rosas IO,et al., “MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis,” PLoS Med.2008;5(4):e93). They are also involved in inflammation and appear to be involved in the pathophysiological process of pulmonary fibrosis (ibid., citing Vij R,Noth I. “Peripheral blood biomarkers in idiopathic pulmonary fibrosis,” Transl Res.2012;159(4):218-27;Dancer RCA,et al., “Metalloproteinases in idiopathic pulmonary fibrosis,” Eur Respir J. 2011;38(6):1461-67).The most studied is MMP-7, which is known to be significantly increased in epithelial cells at both the gene and protein levels and is thought to be active in hyperplastic epithelial cells and alveolar macrophages in IPF (ibid., citing Fujishima S,et al.,“Production and activation of matrix metalloproteinase 7(matrilysin 1) in the lungs of patients with idiopathic pulmonary fibrosis,” Arch Pathol Lab Med. 2010;134(8):1136-42). There is also a significant correlation between high MMP-7 concentrations and disease severity as assessed by forced vital capacity (FVC) and DLCO(%pred) (ibid., citing Rosas IO,et al.,“MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis,” PLoS Med. 2008;5(4):e93). Higher levels were associated with disease progression and decreased survival (>4.3 ng / ml for MMP-7) (Id.). The MMP2 gene provides the instructions to make matrix metallopeptidase 2. This enzyme is produced by cells throughout the body and becomes part of the extracellular matrix, a complex lattice of proteins and other molecules that form in the spaces between cells. One of the main known functions of MMP-2 is to cleave type IV collagen, which is the main structural component of the basement membrane, a thin sheet-like structure that separates and supports cells as part of the extracellular matrix.

[0135] As used herein, the term "microneedling" refers to a cosmetic procedure in which very fine needles puncture the skin, causing controlled damage, inducing the skin to produce more collagen and improving the complexion. Microneedling relies on collagen formation and angiogenesis, which occur as a result of the release of growth factors following needling of the stratum corneum (the outer layer of the skin). This procedure is often used to treat scarring and photoaging.

[0136] The term "nucleic acid" is used herein to refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and, unless otherwise limited, encompasses known analogues which have the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to natural nucleotides (e.g., peptide nucleic acids).

[0137] The term "nucleotide" is used herein to refer to a compound consisting of a heterocyclic base, a sugar, and one or more phosphate groups. In the most common nucleotides, the base is a derivative of a purine or pyrimidine, and the sugar is the pentose deoxyribose or ribose. Nucleotides are monomers of nucleic acids, three or more of which are combined to form nucleic acids. Nucleotides are the structural units of RNA, DNA, and several cofactors, including but not limited to CoA, FAD, DMN, NAD, and NADP. Purines include adenine (A) and guanine (G); pyrimidines include cytosine (C), thymine (T), and uracil (U).

[0138] As used herein, the term "paracrine signaling" refers to short-range cell-cell communication via secreted signal molecules that act on adjacent cells.

[0139] The term "pharmaceutical composition" is used herein to refer to a composition used to prevent, reduce the severity of, cure, or treat a targeted condition or disease. The terms "formulation" and "composition" are used interchangeably herein to refer to the described product of the invention, including all active and inactive ingredients.

[0140] "Pharmaceutically acceptable" is used to refer to a carrier, diluent, or excipient that is compatible with the other ingredients of the formulation or composition and not harmful to the recipient thereof. For example, the term "pharmaceutically acceptable" can mean approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more specifically, in humans.

[0141] The term "primer" refers to a nucleic acid that, when hybridized to a strand of DNA, can initiate the synthesis of an extension product in the presence of a suitable polymerization agent. A primer has a length sufficient to uniquely hybridize to a specific region of a DNA strand. A primer can also be used on an RNA, for example, to synthesize the first strand of cDNA.

[0142] As used herein, the term "purification" and its various grammatical forms refer to a process by which a substance is separated or rendered free from foreign, extraneous, or undesirable elements.

[0143] As used herein, the term "regulating skin condition" includes one or more of inducing increased skin integrity through cell renewal; increasing skin moisture content or humidity; reducing transdermal water loss, peeling, and scaling; improving skin thickness; increasing tensile properties of skin; reducing the appearance of fine lines and wrinkles in the skin; improving skin texture; reducing the size of skin pores; increasing skin smoothness; improving skin blemishes; improving skin tone; or improving the appearance of scars and skin abrasions.

[0144] The term "repair" as used herein as a noun refers to any correction, enhancement, reconditioning, treatment, compensation, normalization, regeneration, mending, patching, etc. that restores function. Used as a verb, it means to correct, enhance, recondition, treat, compensate, normalize, regenerate, repair, patch, or restore function.

[0145] As used herein, the term "skin integrity" refers to intact skin, which is the body's first line of defense against microbial invasion, provides a protective barrier against many environmental threats, and facilitates moisture retention. As used herein, the term "impaired skin integrity" refers to alterations in the epidermis and / or dermis such that the skin is damaged, susceptible to damage, or unable to heal normally.

[0146] The term "stem cell" refers to an undifferentiated cell with self-renewal potential that can generate daughter cells that have high proliferation potential and can terminally differentiate into multiple different cell phenotypes. As used herein, the term "renewal" or "self-renewal" refers to the process by which stem cells divide to generate one (asymmetric division) or two (symmetric division) daughter cells that have developmental potential that is indistinguishable from that of the mother cell. Self-renewal includes both proliferation and maintenance of an undifferentiated state.

[0147] The term "adult (somatic) stem cells" as used herein refers to undifferentiated cells found among differentiated cells in tissues or organs. Their main role in vivo is to maintain and repair the tissues in which they are found. Adult stem cells, which have been identified in many organs and tissues, including the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, gastrointestinal tract, liver, ovarian epithelium, and testes, are thought to reside in specific regions of each tissue, known as stem cell niches, where they may remain quiescent (non-dividing) for long periods of time until activated by the normal need for more cells to maintain the tissue, or by disease or tissue damage.

[0148] As used herein, the term "symptom" refers to a sign or manifestation of a disorder or disease, especially when experienced by an individual as an alteration from normal function, sensation, or appearance.

[0149] As used herein, the term "therapeutic agent" or "active agent" refers to an ingredient, component or constituent of a composition of the described invention that is responsible for the intended therapeutic effect.

[0150] As used herein, the term "therapeutic component" refers to a therapeutically effective dose (i.e., dosage and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease symptom in a percentage of the population. An example of a commonly used therapeutic component is the ED50, which represents the amount administered at a particular dose that is therapeutically effective against a particular disease symptom in 50% of the population.

[0151] As used herein, the term "therapeutic effect" refers to an outcome of treatment that is deemed desirable and beneficial. Therapeutic effect may include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. Therapeutic effect may also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease symptoms.

[0152] As used herein, the term "tissue" refers to a collection of similar cells and the intercellular material that surrounds them. For example, adipose tissue is a connective tissue that is mainly composed of adipocytes surrounded by reticular fibers and arranged in groups of lobules or along the pathways of smaller blood vessels. Connective tissue is the supporting or skeletal tissue of the body that is formed of fibrous material and ground substance with a large number of cells of various types. It originates from mesenchyme, which in turn originates from mesoderm. Types of connective tissue include, but are not limited to, loose or loose connective tissue; adipose connective tissue; dense connective tissue, defined or nondefined connective tissue, white fibrous connective tissue; elastic connective tissue; gelatinous connective tissue; lymphatic tissue; cartilaginous connective tissue and osseous connective tissue.

[0153] As used herein, the terms "treat", "treated" or "treating" refer to both therapeutic treatment and / or prophylactic or preventative measures, the purpose being to prevent or slow (alleviate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desirable clinical outcome. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms; attenuation of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delay in onset or slowing of progression of the condition, disorder or disease; improvement of the condition, disorder or disease state; and remission (partial or total), or enhancement or amelioration of the condition, disorder or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. "Treatment" can also mean prolonging survival compared to the expected survival in the absence of treatment.

[0154] Preparation of EVs and EVs According to some embodiments, the described invention provides a composition comprising a population of membrane (i.e., lipid bilayer) vesicles (EVs) derived from amniotic fluid. According to some embodiments, the EVs are derived from amniotic fluid mesenchymal stem cells (MSCs). When included in a pharmaceutical composition, the pharmaceutical composition comprises a composition comprising a population of isolated EVs and a pharma- ceutically acceptable carrier. According to some embodiments, the amniotic fluid is allogeneic with respect to the subject to whom the pharmaceutical composition is intended to be administered. According to some embodiments, the amniotic fluid is autologous with respect to the subject to whom the pharmaceutical composition is intended to be administered. According to some embodiments, the amniotic fluid is mammalian amniotic fluid. According to some embodiments, the amniotic fluid is human amniotic fluid.

[0155] amniotic fluid Amniotic fluid samples are obtained by amniocentesis performed between 16 and 20 weeks of gestation for fetal karyotype analysis. A two-step culture protocol can be used to isolate MSCs from amniotic fluid (Tsai MS, et al., Hum Reprod. 2004 Jun;19(6):1450-6). For amniotic cell culture (first step), in situ primary cultures are set up in tissue culture grade dishes (Irvine Scientific, Santa Ana, CA) using Chang's medium. Selection of metaphases and colony determination are based on the basic requirements for prenatal cytogenetic diagnosis in amniotic cells (Moertel CA, et al., 1992; Prenat Diagn 12,671-683). For MSC culture (second step), non-adherent amniotic fluid cells in the supernatant medium were collected 5 days after primary culture of amniotic cells and continued until fetal chromosome analysis was completed. The cells were then centrifuged and separated by 25 cm 2 The cells were plated in 5 ml of alpha modified minimal essential medium (α-MEM; Gibco-BRL) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, UT) and 4 ng / ml basic fibroblast growth factor (bFGF; R&D systems, Minneapolis, MN) in a flask and incubated at 37°C in 5% humidified CO2 for MSC culture. Similar to MSCs derived from umbilical cord blood and early fetal tissues, surface antigens such as SH3, SH4, CD29, CD44, and HLA-A, B, C (MHC class I) can be detected, while CD10, CD11b, CD14, CD34, CD117, HLA-DR, DP, DQ (MHC class II) and EMA are absent (Tsai MS, et al., Hum Reprod. 2004 Jun;19(6):1450-6; Pittenger MF, et al., Science 284,143-7; Colter DC, et al., Proc Natl Acad Sci USA 98,78415; Young HY, et al., Anat Rec 264,51-62).

[0156] According to some embodiments, the EVs comprise microvesicles, exosomes, or both. According to some embodiments, the EVs are between about 30 nm and 200 nm, i.e., at least 30 nm, at least 31 nm, at least 32 nm, at least 33 nm, at least 34 nm, at least 35 nm, at least 36 nm, at least 37 nm, at least 38 nm, at least 39 nm, at least 40 nm, at least 41 nm, at least 42 nm, at least 43 nm, at least 44 nm, at least 45 nm, at least 46 nm, at least 47 nm, at least 48 nm, at least 49 nm, at least 50 nm, at least 51 nm, at least 52 nm, at least 53 nm, at least 54 nm, at least 55 nm, at least 56 nm, at least 57 nm, at least 58 nm, at least 59 nm, at least 60 nm, at least 61 nm, at least 62 nm, at least 63 nm, at least 64 nm, at least 65 nm, at least 66 nm, at least 67 nm, at least 68 nm, at least 69 nm, at least 70 nm, at least 71 nm, at least 72 nm, at least 73 nm, at least at least 74 nm, at least 75 nm, at least 76 nm, at least 77 nm, at least 78 nm, at least 79 nm, at least 80 nm, at least 81 nm, at least 82 nm, at least 83 nm, at least 84 nm, at least 85 nm, at least 86 nm, at least 87 nm, at least 88 nm, at least 89 nm, at least 90 nm, at least 91 nm, at least 92 nm, at least 93 nm, at least 94 nm, at least 95 nm, at least 96 nm, at least 97 nm, at least 98 nm, at least 99 nm, at least 100 nm, at least 101 nm, at least 102 nm, at least 103 nm, at least 104 nm, at least 105 nm, at least 106 nm, at least 107 nm, at least 108 nm, at least 109 nm, at least 110 nm, at least 120 nm, at least 121 nm, at least 122 nm, at least 123 nm, at least 124 nm, at least 125 nm, at least 126 nm, at least 127 nm, at least 128 nm,at least 129 nm, at least 130 nm, at least 131 nm, at least 132 nm, at least 133 nm, at least 134 nm, at least 135 nm, at least 136 nm, at least 137 nm, at least 138 nm, at least 139 nm, at least 140 nm, at least 141 nm, at least 142 nm, at least 143 nm, at least 144 nm, at least 145 nm, at least 146 nm, at least 147 nm, at least 148 nm, at least 149 nm, at least 150 nm, at least 151 nm, at least 152 nm, at least 153 nm, at least 154 nm, at least 155 nm, at least 156 nm, at least 157 nm, at least 158 ​​nm, at least 159 nm, at least 160 nm, at least 161 nm, at least 162 nm, at least 163 nm, at least 164 nm, at least 1 65 nm, at least 166 nm, at least 167 nm, at least 168 nm, at least 169 nm, at least 170 nm, at least 171 nm, at least 172 nm, at least 173 nm, at least 174 nm, at least 175 nm, at least 176 nm, at least 177 nm, at least 178 nm, at least 179 nm, at least 180 nm, at least 181 nm, at least 182 nm, at least 183 nm, at least 184 nm, at least 185 nm, at least 186 nm, at least 187 nm, at least 188 nm, at least 189 nm, at least 190 nm, at least 191 nm, at least 192 nm, at least 193 nm, at least 194 nm, at least 195 nm, at least 196 nm, at least 197 nm, at least 198 nm, at least 199 nm, or at least 200 nm. According to some embodiments, EVs have a diameter ranging from about 50 nm to about 200 nm, i.e., at least 50 nm, at least 51 nm, at least 52 nm, at least 53 nm, at least 54 nm, at least 55 nm, at least 56 nm, at least 57 nm, at least 58 nm, at least 59 nm, at least 60 nm, at least 61 nm, at least 62 nm, at least 63 nm, at least 64 nm,at least 65nm, at least 66nm, at least 67nm, at least 68nm, at least 69nm, at least 70nm, at least 71nm, at least 72nm, at least 73nm, at least 74nm, at least 75nm, at least 76nm, at least 77nm, at least 78nm, at least 79nm, at least 80nm, at least 81nm, at least 82nm, at least 83nm, at least 84nm, at least 85nm, at least 86nm, at least 87nm, at least 88nm, at least 89nm , at least 90 nm, at least 91 nm, at least 92 nm, at least 93 nm, at least 94 nm, at least 95 nm, at least 96 nm, at least 97 nm, at least 98 nm, at least 99 nm, at least 100 nm, at least 101 nm, at least 102 nm, at least 103 nm, at least 104 nm, at least 105 nm, at least 106 nm, at least 107 nm, at least 108 nm, at least 109 nm, at least 110 nm, at least 120 nm, at least 121 nm, at least 1 22nm, at least 123nm, at least 124nm, at least 125nm, at least 126nm, at least 127nm, at least 128nm, at least 129nm, at least 130nm, at least 131nm, at least 132nm, at least 133nm, at least 134nm, at least 135nm, at least 136nm, at least 137nm, at least 138nm, at least 139nm, at least 140nm, at least 141nm, at least 142nm, at least 143nm, at least 144nm, at least At least 145 nm, at least 146 nm, at least 147 nm, at least 148 nm, at least 149 nm, at least 150 nm, at least 151 nm, at least 152 nm, at least 153 nm, at least 154 nm, at least 155 nm, at least 156 nm, at least 157 nm, at least 158 ​​nm, at least 159 nm, at least 160 nm, at least 161 nm, at least 162 nm, at least 163 nm, at least 164 nm, at least 165 nm, at least 166 nm, at least 167 nm,at least 168 nm, at least 169 nm, at least 170 nm, at least 171 nm, at least 172 nm, at least 173 nm, at least 174 nm, at least 175 nm, at least 176 nm, at least 177 nm, at least 178 nm, at least 179 nm, at least 180 nm, at least 181 nm, at least 182 nm, at least 183 nm, at least 184 nm, at least 185 nm, at least 186 nm, at least 187 nm, at least 188 nm, at least 189 nm, at least 190 nm, at least 191 nm, at least 192 nm, at least 193 nm, at least 194 nm, at least 195 nm, at least 196 nm, at least 197 nm, at least 198 nm, at least 199 nm, or at least 200 nm. According to some embodiments, by electron microscopy, the EVs appear to have a cup-shaped morphology. According to some embodiments, EVs sediment at about 100,000 x g and have a buoyant density of about 1.10 to about 1.21 g / ml in sucrose.

[0157] According to several embodiments, EVs contain proteins, nucleic acids, or both, including RNA species such as miRNA.

[0158] According to some embodiments, the extracellular vesicles are isolated EVs. As used herein, the term "population of isolated EVs" refers to a population of EVs that is physically separated from its natural environment. According to some embodiments, the population of isolated EVs can be physically separated, in whole or in part, from the tissue or cell in which it naturally occurs. According to some embodiments, the composition comprising isolated EVs may be substantially free of cells or cellular components, or free of or substantially free of conditioned medium. According to some embodiments, the concentration of isolated EVs may be higher than the concentration of EVs present in unmanipulated conditioned medium. According to some embodiments, the population of EVs comprises an enriched subpopulation of EVs that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.

[0159] According to some embodiments, EVs can be isolated from the conditioned medium and recovered from the cultured MSCs, including metabolites, growth factors, RNA and proteins released into the medium by the cultured MSCs.

[0160] According to some embodiments, the method for recovery of EVs from MSCs includes first culturing MSCs under standard conditions until they reach about 70% confluency, then culturing the cells in serum-free medium for 24 hours. The conditioned medium is then collected and subjected to differential centrifugation at 400×g for 10 min and 12000×g for 10 min to remove whole cells and cell debris, generating clarified conditioned medium. The clarified conditioned medium is then concentrated by ultrafiltration using a 100 kDa MWCO filter (Millipore) and then centrifuged again at 12000×g for 10 min. EVs are then isolated using size exclusion chromatography by loading the concentrated clarified conditioned medium onto a PBS-equilibrated Chroma S-200 column (Clontech), eluting with PBS, and collecting fractions of 350-550 μl. Fractions containing EVs are identified and optionally pooled. Protein concentration is measured using the standard Bradford method (Bio-Rad). Aliquots of the concentrated extracellular vesicle preparation are stored at -80°C.

[0161] According to some embodiments, EVs can also be purified by ultracentrifugation of the clarified conditioned medium at 100,000×g. According to some embodiments, they can also be purified by ultracentrifugation into a sucrose cushion. A GMP method for EV purification from dendritic cells is described in J Immunol Methods. 2002;270:211-226, which is incorporated herein by reference.

[0162] According to some embodiments, EVs can be purified by differential filtration through nylon membrane filters of defined pore sizes. For example, initial filtration through larger pore sizes retains cellular fragments and debris; subsequent filtration through smaller pore sizes retains the EVs and purifies them from smaller sized contaminants.

[0163] Treatment method According to some embodiments, a method of promoting wound healing in a subject in need thereof comprises contacting wound tissue of the subject with a first composition comprising a therapeutic amount of extracellular vesicles (EVs) derived from human amniotic fluid (AF), the therapeutic amount being effective to reduce wound area and promote repair of the wound tissue. According to some embodiments, the EVs are derived from amniotic fluid mesenchymal stem cells (MSCs).

[0164] A "therapeutically effective amount," "therapeutic amount," or "effective amount" of a pharmaceutical composition comprising the EV of the described invention is a predetermined amount calculated to achieve a desired biological effect. The activities contemplated by the described methods include both medical and / or prophylactic treatment, as appropriate. The specific dose of the composition administered according to the described invention to obtain a therapeutic and / or prophylactic treatment effect will, of course, be determined by the particular circumstances surrounding the case, including, for example, the composition administered, the route of administration, and the pathology to be treated. According to some embodiments, a standard effective amount of a pharmaceutical composition is about 1×10 5 ~Approx. 1×10 9 MSCs, i.e., 1 x 10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×107 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , or 1 × 10 9 The therapeutically effective amount of the composition of the present invention is generally sufficient to achieve an effective systemic or localized concentration in tissues when administered in a physiologically acceptable excipient composition.

[0165] According to some embodiments, the composition is effective for promoting wound healing by activating epithelial cells to transform into a mesenchymal cell phenotype (EMT). According to some embodiments, the composition is effective for increasing mRNA levels of one or more of vimentin, N-cadherin, Col1a1, Acta2, or TGFbr2. According to some embodiments, the method further comprises measuring the levels of one or more of vimentin, N-cadherin, Col1a1, Acta2, or TGFbr2.

[0166] According to some embodiments, the contacting is performed topically, subcutaneously, intranasally, intratracheally, orally, parenterally, intravenously, or intraperitoneally. As used herein, the term "parenteral" refers to introduction into the body by a means other than the digestive tract, for example, but not limited to, by injection (i.e., administration by injection), for example, including subcutaneous (i.e., injection under the skin), intramuscular (i.e., injection into a muscle), intravenous (i.e., injection into a vein), or infusion techniques. According to some embodiments, the contacting is performed topically or subcutaneously.

[0167] According to some embodiments, the subject is a human patient who has been diagnosed with a wound or is symptomatic of a wound. According to some embodiments, the subject is a human patient who has been diagnosed with a wound or is symptomatic of a chronic wound. According to some embodiments, the subject is a human patient who has been diagnosed with or is at risk of developing a wound into a chronic wound. According to some embodiments, the subject is a human patient who has been diagnosed with or is symptomatic of a diabetic ulcer, a pressure ulcer, or a venous ulcer. According to some embodiments, the subject is a human patient who has been diagnosed with or is symptomatic of a burn.

[0168] According to some embodiments, the method further comprises contacting the wound tissue of the subject with a second composition comprising a therapeutic amount of EV-depleted AF, the therapeutic amount of the second composition being effective to activate mesenchymal-epithelial transition (MET) and promote repair of the wound tissue. According to some embodiments, the length of time between contacting the tissue with the first composition and the second composition is about 4 to about 24 hours, i.e., about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. According to some embodiments, the length of time between contacting the tissue with the first composition and the second composition is more than 24 hours. According to some embodiments, the second composition is effective to increase Stat3 mRNA levels. STAT3 (signal transducer and activator of transcription 3) is a transcription factor that regulates the expression of genes involved in late stage wound healing, such as epithelial cell proliferation, extracellular matrix remodeling, angiogenesis, and suppression of inflammation. According to some embodiments, the method further comprises measuring the level of Stat3 mRNA.

[0169] According to some embodiments, EVs are purified from amniotic fluid by one or more of: a) ultracentrifugation; b) sucrose density gradient centrifugation; c) column chromatography; d) size exclusion; or e) filtration through a device containing an affinity matrix selective for EVs. According to some embodiments, EVs are further filtered by size. According to some embodiments, EVs are characterized by an average diameter of about 50 nm to about 200 nm. According to some embodiments, EVs are characterized by an average diameter of about 50 nm to about 1000 nm.

[0170] According to some embodiments, a two-step method of promoting wound healing in a subject in need thereof includes, in order: a. contacting a wound with a composition comprising extracellular vesicles (EVs) derived from amniotic fluid (AF) to promote early stage wound healing in the subject; and b. contacting a wound with a composition comprising EV-depleted AF to promote late stage wound healing in the subject. According to some embodiments, early stage wound healing is characterized by activating epithelial-mesenchymal transition (EMT) and inducing cell migration, and late stage wound healing is characterized by activating mesenchymal-epithelial transition (MET) and epithelialization of the wound. According to some embodiments, the EVs are derived from amniotic fluid mesenchymal stem cells (MSCs). According to some embodiments, the EVs are characterized by a sedimentation at about 100,000×g, a buoyant density in sucrose of about 1.10-1.21 g / ml, and an average diameter of about 50 nm to about 200 nm. According to some embodiments, the contacting is topical or subcutaneous.

[0171] According to some embodiments, a method for regulating skin condition in a subject in need thereof includes contacting the skin of the subject with a composition comprising a therapeutic amount of extracellular vesicles (EVs) derived from human amniotic fluid (AF), the therapeutic amount being effective to improve skin texture, reduce wrinkles, or both, thereby regulating skin condition. According to some embodiments, the method further includes microneedling the skin prior to contacting with the composition. According to some embodiments, the composition is effective to regulate skin condition by activating epithelial-mesenchymal transition (EMT).

[0172] formulation According to some embodiments, the composition further comprises a pharma- ceutically acceptable carrier. The phrase "pharmaceutically acceptable carrier" is art-recognized. The phrase is used to mean a substantially non-toxic carrier that can be conventionally used for the administration of pharmaceutical agents, in which the isolated exosomes of the present invention are stable and maintain bioavailability. A pharma- ceutically acceptable carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the mammal to be treated. In addition, the stability and bioavailability of the active agent should be maintained. A pharma- ceutically acceptable carrier can be liquid or solid, and is selected in view of the proposed method of administration to provide the desired volume, concentration, etc., when combined with the active agent and other components of a given composition. Exemplary carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating substances involved in carrying or transporting the agent of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other components of the formulation and not harmful to the patient. Some examples of substances which may serve as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances used in pharmaceutical formulations.Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin, which is incorporated herein by reference in its entirety. According to some embodiments, the pharma- ceutically acceptable carrier is sterile, pyrogen-free water. According to some embodiments, the pharma-ceutically acceptable carrier is Ringer's lactate, sometimes known as lactated Ringer's solution.

[0173] Wetting agents, emulsifying agents and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0174] Examples of pharma- ceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0175] Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, tragacanth, gelatin, syrup, methylcellulose, methyl and propyl hydroxybenzoate, talc, magnesium stearate, water and mineral oil.The formulation may further include lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweeteners or flavoring agents.The composition may be formulated to release active ingredient quickly, sustained or delayed after administration to a patient using procedures well known in the art.

[0176] The local delivery of therapeutic amounts of compositions for the treatment of lung injury or fibrotic lung disease can be achieved by various techniques that administer compounds to or near the target site.The examples of local delivery techniques are not intended to be limiting, but are intended to illustrate available techniques.Examples include local delivery catheters, site-specific carriers, implants, direct injection, or direct application, such as topical application, and administration by inhalation in the case of the lungs.

[0177] Localized delivery by implant refers to the surgical placement of a matrix containing a pharmaceutical agent at the affected area. The implanted matrix releases the pharmaceutical agent by diffusion, chemical reaction, or solvent activators.

[0178] The specific mode of administration varies depending on the indication. The selection of the specific route of administration and the administration schedule should be adjusted or titrated by the clinician according to methods known to the clinician to obtain the optimal clinical response. The amount of the active agent administered is an amount sufficient to provide the intended therapeutic effect. The dosage administered depends on the characteristics of the subject to be treated, such as the specific mammal or human being to be treated, age, weight, health, type of concurrent treatment, if any, and frequency of treatment, and can be easily determined by those skilled in the art (e.g., by a clinician).

[0179] Pharmaceutical formulations containing the described active agents of the present invention and suitable carriers may be solid dosage forms, including but not limited to tablets, capsules, cachets, pellets, pills, powders and granules, containing an effective amount of the described polymer or copolymer of the present invention; topical dosage forms, including but not limited to solutions, powders, liquid emulsions, liquid suspensions, semisolids, ointments, pastes, creams, gels, jellies and foams; and parenteral dosage forms, including but not limited to solutions, suspensions, emulsions and dry powders. It is also known in the art that the active ingredient may be included in such formulations together with pharma- ceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water-soluble vehicles, emulsifiers, buffers, wetting agents, humectants, solubilizers, and preservatives. Means and methods of administration are known in the art, and those skilled in the art can refer to various pharmacological references for guidance. See, e.g., Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980).

[0180] The pharmaceutical composition of the present invention described can be formulated for parenteral administration, for example, by injection, for example, bolus injection or continuous infusion. The pharmaceutical composition can be administered by continuous subcutaneous infusion over a period of time. The preparation for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of a preservative. The pharmaceutical composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulatory agents, such as suspending, stabilizing, and / or dispersing agents.

[0181] For oral administration, pharmaceutical compositions can be easily formulated by combining the active agent(s) with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the active substances of the present disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by the patient to be treated. Medicaments for oral use can be obtained by adding solid excipients, optionally grinding the resulting mixture, processing the granular mixture, and optionally adding suitable excipients to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including but not limited to lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, including, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0182] Dragee cores can be provided with suitable coatings.For this purpose, can be used optionally gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and concentrated sugar solutions containing suitable organic solvents or solvent mixtures.Dyes or pigments can be added to tablets or dragee coatings to identify or characterize different combinations of active compound doses.

[0183] Other pharmaceutical preparations that can be used orally include, but are not limited to, push-fit capsules made of gelatin, and soft, sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers can be added. All preparations for oral administration should be in a dosage suitable for such administration.

[0184] For buccal administration, the compositions may take the form of, for example, tablets or lozenges formulated in conventional manner.

[0185] For administration by inhalation, the compositions for use according to the invention described herein can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0186] In addition to the formulations described previously, the compositions of the invention described can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.

[0187] Depot injections can be administered at intervals of about 1 to about 6 months or more.Thus, for example, the compositions can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0188] Pharmaceutical compositions containing any one or more active agents disclosed herein may also include suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0189] For parenteral administration, the pharmaceutical composition can be formulated, for example, as a solution, suspension, emulsion, or lyophilized powder with a pharma- ceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous media such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.

[0190] The invention described relates to all routes of administration, including subcutaneous, topical, intramuscular, sublingual, intravenous, intraperitoneal, intranasal, intratracheal, intradermal, intramucosal, intracavernous, rectal, intraantral, gastrointestinal, intraductal, intrathecal, intraventricular, intrapulmonary, intraabscess, intraarticular, subpericardial, intraaxillary, intrapleural, intradermal, buccal, transmucosal, transdermal, inhalation, and nebulizer. Alternatively, the pharmaceutical composition may be introduced into the cells to be removed from the individual by various means, including, for example, particle bombardment via liposomes or other nanoparticle devices.

[0191] According to some embodiments, the pharmaceutical compositions of the claimed invention contain one or more therapeutic agents other than EV, as described. Examples of such additional active therapeutic agents include one or more analgesics, anti-inflammatory agents, or antibacterial agents.

[0192] Examples of painkillers include codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, and fentanyl.

[0193] Examples of anti-inflammatory agents include aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolacnabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sulindac, and tolmetin.

[0194] Examples of antimicrobial agents include, but are not limited to, antibiotics such as, for example, bacitracin, mafenide, mupirocin, neomycin, silver sulfadiazine, curcumin, and honey; and preservatives such as, for example, biguanides, silver, iodine, and chlorine compounds.

[0195] According to the above embodiments, the pharmaceutical composition may be administered once for a predetermined period of time or as a long-term maintenance therapy, for example, until the condition improves, is cured, or for the duration of the subject's life. The predetermined period of time may be 1 week, 2 weeks, 3 weeks, 4 weeks, and up to 1 year (including any period between such values, including the endpoints). According to some embodiments, the pharmaceutical composition may be administered for about 1 day, about 3 days, about 1 week, about 10 days, about 2 weeks, about 18 days, about 3 weeks, or any range between any of these values, including the endpoints. According to some embodiments, the pharmaceutical composition may be administered for more than 1 year, about 2 years, about 3 years, about 4 years, or more.

[0196] According to the foregoing embodiments, the composition or pharmaceutical composition may be administered less than once a day (eg, every other day), once a day, twice a day, three times a day, four times a day or more.

[0197] All journal articles, patents, and other publications referenced are incorporated by reference in their entirety.

[0198] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed herein, subject to any specific excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0199] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In carrying out or testing the present invention, any method and material similar or equivalent to the methods and materials described herein can be used, but exemplary methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which they are cited. EXAMPLES

[0200] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of the invention as contemplated by the inventors, nor are they intended to represent that the following experiments are all or the only experiments performed.

[0201] Example 1: Amniotic fluid-derived exosomes promote wound healing by activating epithelial-mesenchymal transition Both extracellular vesicles (EV) / exosome-based products and human amniotic fluid (AF) have attracted considerable attention for their potential applications in regenerative medicine. However, the mechanism of action by which they exert their therapeutic effect on target cell types remains unclear. Herein, we tested the hypothesis that amniotic fluid exosomes are required to promote wound healing modeled in vitro.

[0202] Epithelial-mesenchymal transition (EMT) is a biological process that allows polarized epithelial cells, which normally interact with the basement membrane via their basal surface, to undergo multiple biochemical changes and thereby adopt a mesenchymal cell phenotype, including enhanced migratory capacity, invasiveness, increased resistance to apoptosis, and a greatly increased production of ECM components (Kalluri, R. and Weinberg, RA, J. Clin. Invest. (2009) 119:1420-1428, citing Kalluri R., Neilson EGJ Clin. Invest. 2003;112:1776-1784). Completion of EMT is marked by the degradation of the underlying basement membrane and the formation of mesenchymal cells capable of migrating away from the epithelial layer in which they originate (ibid.).

[0203] EMT, associated with implantation, embryogenesis, and organ development, is orchestrated to generate diverse cell types that share a common mesenchymal phenotype. This class of EMT ("Type 1 EMT") does not cause fibrosis and does not induce an invasive phenotype that leads to systemic spread via the circulation (Ibid., citing Zeisberg M., Neilson EGJ Clin. Invest. 2009;119:1429-1437). Among other outcomes, these Type 1 EMTs can generate mesenchymal cells (primary mesenchyme), which can then undergo mesenchymal-epithelial transition (MET), converting mesenchymal cells into epithelial derivatives to generate secondary epithelium) (Ibid.).

[0204] Type 2 EMT is associated with wound healing, tissue regeneration, and organ fibrosis (ibid.). In type 2 EMT, a program is initiated as part of repair-related events that normally generate fibroblasts and other related cells to remodel tissue after trauma or inflammatory damage. However, in contrast to type 1 EMT, these type 2 EMTs are associated with inflammation and are terminated when inflammation subsides, as observed during wound healing and tissue regeneration. In the context of organ fibrosis, type 2 EMT continues to respond to ongoing inflammation, which may ultimately result in organ destruction. Tissue fibrosis is essentially an unabated form of wound healing due to persistent inflammation (ibid.).

[0205] Type 3 EMT occurs in tumor cells that have previously undergone genetic and epigenetic changes, particularly in genes that favor clonal expansion and localized tumor development. These changes, particularly affecting oncogenes and tumor suppressor genes, cooperate with EMT regulatory circuits to produce outcomes that differ significantly from those observed in the other two types of EMT. Cancer cells undergoing type 3 EMT can invade and metastasize, thereby causing the final life-threatening symptoms of cancer progression. Importantly, cancer cells can go through EMT to various degrees, with some cells acquiring some mesenchymal traits while retaining many epithelial traits, and others removing all traces of their epithelial origin and becoming completely mesenchymal. It is still unclear which specific signals induce type 3 EMT in cancer cells (ibid.).

[0206] Several different molecular processes are involved in initiating EMT and allowing it to reach completion. These include the activation of transcription factors, the expression of specific cell surface proteins, the reorganization and expression of cytoskeletal proteins, the production of ECM degrading enzymes, and changes in the expression of specific microRNAs. In many cases, the factors involved are also used as biomarkers to indicate the passage of cells through EMT (ibid.).

[0207] The reverse process, mesenchymal-epithelial transition (MET), can generate epithelial cells as well. MET events are defined as events in which mesenchymal cells lose motile migratory properties and acquire cell polarity and adhesion to epithelia. Both MET and EMT occur in normal tissues, including gastrulation and regenerating tissues, as well as in abnormal tissues of fibrous organs or tumors (Li B,et al. PLoS One. 2011;6(2):e17092,Kalluri R,Weinberg RA. The Journal of clinical investigation. 2009;119:1420;citing Polyak K,Weinberg RA. Nat Rev Cancer. 2009;9:265-273). Thus, there is a strong relationship between EMT / MET and stem cells. Indeed, EMT drives mammary epithelial cells to dedifferentiate into mesenchymal-like mammary stem cells and cancer stem cells (ibid., citing Mani SA,et al. Cell. 2008;133:704-715). Furthermore, induced pluripotent stem cells (iPSCs) are derived from mouse embryonic fibroblasts (MEFs) by MET during the early stage of reprogramming (ibid., citing Polo JM,Hochedlinger K. Cell Stem Cell. 2010;7:5-6;Li R,et al. Cell Stem Cell. 2010;7:51-63;Samavarchi-Tehrani P,et al. Cell Stem Cell. 2010;7:64-77). These results suggest that MET may be related to stem cell activity.

[0208] To identify EMT / MET, vimentin has been widely applied as a mesenchymal indicator (ibid., Kalluri R, Weinberg RA. The Journal of clinical investigation. 2009;119:1420; Mani SA,et al. Cell. 2008;133:704-715; Arias AM. Cell. 2001;105:425-431; Thiery JP,et al. Cell. 2009;139:871-890; Gershengorn MC,et al. Science. 2004;306:2261-2264; Thiery JP. Nature Reviews Cancer. 2002;2:442-454). Vimentin is an intermediate filament protein functionally involved in maintaining the structure of mesenchymal cells (ibid., citing Stenger AM, et al. Molecular Brain Research. 1992;13:273-275). In addition to being associated with mesenchymal cell migration and proliferation, vimentin is an indicator of changes in cell morphology or cytoskeletal rearrangements (ibid., citing Venetianer A, et al. Nature. 1983;305:730-733; Hedberg KK, Chen LB. Experimental cell research. 1986;163:509-517). During mouse embryonic gastrulation, vimentin increases in fibroblasts that undergo lamellar division through the primitive streak to become mesoderm (Id., Eckes B, et al. J Cell Sci. 2000;113(Pt13):2455-2462; Lane EB, et al. Nature. 1983;303:701-704; Franke WW, et al. Differentiation. 1982;23:43-59), indicating that vimentin plays a role in cell transformation and tissue organization.Furthermore, vimentin is closely associated with loss of plasma membrane polarity in fibroblasts (ibid. citing Oriolo AS, et al. Experimental cell research. 2007;313:2255-2264), and cell adhesion and polarization are associated with a decrease in vimentin (ibid. citing Nieminen M, et al. Nature cell biology. 2006;8:156-162).

[0209] Whereas EMT is associated with the reactivation or reprogramming of epithelial cells, MET appears to drive stem cells to a quiescent state (ibid. Mani SA,et al. Cell. 2008;133:704-715; citing Spaderna S,et al. Verh Dtsch Ges Pathol. 2007;91:21-28). MET is also involved in the inactivation of other cells, for example, in wound healing, activated fibroblasts lose cell polarity, migrate to the wound site, and differentiate into keratinocytes (ibid. Eckes B,et al. J Cell Sci. 2000;113(Pt13):2455-2462), a process driven by MET.

[0210] method Amniotic fluid (AF) acquisition and processing. AF was donated from full-term elective cesarean sections that were screened and found negative for infection, or was donated for research use with Dermacyte™ (purified AF) by Merakris Therapeutics. Donor AF was subjected to sequential centrifugation at 4°C, then passed through a 0.2 μm filter, and either used immediately or stored at -80°C. AF exosomes were purified using the ExoQuick TC-ULTRA kit (SBI Biosciences) according to the manufacturer's specifications, filtered from Dermacyte Liquid in the size range of 50-200 nm, and then quantitative and qualitative analysis was performed using a ZetaSizer (Malvern Panalytical).

[0211] Cell culture and scratch test assay. Mouse C2C12 myoblasts and MMM fibroblasts were routinely cultured in DMEM + 10% FBS with 1% penicillin / streptomycin (complete medium) on standard TC-treated Corning plasticware. Scratch test assays were performed by growing C2C12 or MMM cells in complete medium to approximately 70-90% confluence and then adding the medium type of interest, e.g., serum-free medium (SFM; consisting of 50% IMDM (Gibco), 50% F12 (Gibco), 1 mg / ml polyvinyl alcohol (Sigma), 1% chemically defined lipid concentrate (Gibco), 450 μM monothioglycerol (Sigma)) alone, SFM + 10% AF, SFM + AF extracellular vesicles (EVs) / exosomes, or SFM + 10% EVs / exosomes-depleted AF. After cells were equilibrated for 2 h in each medium type of interest, vertical scratches were created with a standard micropipette tip of 200 μl size. Dissociated cells were aspirated from the wells and the medium was replaced. Photographs were recorded at each time point by drawing a horizontal line at the bottom of each well as a reference point and taking pictures of the scratch above or below the horizontal line (or both) consistently over time using a bright-field microscope with a 20x objective. The area of ​​the scratch was determined by measuring pixel counts with ImageJ software and plotted as the mean measurement of absolute pixel count or area relative to the scratch at time zero, with error bars indicating standard deviation. Statistical significance was tested using a Student's t-test.

[0212] RNA extraction and RT-qPCR. 24 hours after performing the scratch test assay in C2C12 myoblasts, cells were lysed and RNA was extracted (ReliaPrep, Promega) and quantified using a NanoDrop (ThermoScientific). 100 ng of total RNA was used for reverse transcription with SuperScriptIII enzyme (ThermoScientific), the resulting cDNA was then diluted 5-10× and 2 μL was used as input for running 20 μl qPCR reactions for 40 cycles with 2× SYBR Power MasterMix (Applied Biosystems) on an ABI StepOnePlus qPCR thermal cycler (Applied Biosystems). 2 -ΔΔCt The RNA abundance for the housekeeping gene Hmbs was determined using the method. The specificity of the product was confirmed by melting curve analysis. The primer sequences are as follows: mHmbs_qF 5'-CAGAGAAAGTTCCCCCACCT-3' (SEQ ID NO: 1) mHmbs_qR 5'-AATTCCTGCAGCTCATCCAG-3' (SEQ ID NO: 2) mVimentin_qF 5'-AAACGAGTACCGGAGACAGGT-3' (SEQ ID NO: 3) mVimentin_qR 5'-TCTCTTCCATCTCACGCATCT-3' (SEQ ID NO: 4) mCol1a1_qF 5'-GCCAAGAAGACATCCCTGAA-3' (SEQ ID NO:5) mCol1a1_qR 5'-CAGATCAAGCATACCTCGGG-3' (SEQ ID NO: 6) mTgfbr2_qF 5'-TGGACCCTACTCTGTCTGTGG-3' (SEQ ID NO: 7) mTgfbr2_qR 5'-ACTCCACGTTTTCCAGATTCA-3' (SEQ ID NO: 8) mActa2_qF 5'-ACTGGGACGACATGGAAAAG-3' (SEQ ID NO: 9) mActa2_qR 5'-GTTCAGTGGTGCCTCTGTCA-3' (SEQ ID NO: 10) mN-Cad_qF 5'-GGACATCATCACTGTGGCAG-3' (SEQ ID NO: 11) mN-Cad_qR 5'-TTCCATGTCTGTGGCTTGAA-3' (SEQ ID NO: 12) mE-Cad_qF 5'-CCGGGACTCCAGTCATAGG-3' (SEQ ID NO: 13) mE-Cad_qR 5'-CAGCTCTGGGTTGGATTCAG-3' (SEQ ID NO: 14)

[0213] result Figure 1 shows representative brightfield microscopy images of C2C12 myoblasts during a scratch test wound healing assay at 0, 12, 18, and 24 h, incubated with unconditioned serum-free medium + 10% amniotic fluid (uncSFM+AF), uncSFM containing the same amount of amniotic fluid-derived exosomes as in uncSFM+10% AF (uncSFM+AFexos), or uncSFM+10% exosome-depleted amniotic fluid (uncSFM+exo(-)AF). The results of this assay are quantified in Figure 2. uncSFM with 10% exosome / EV-depleted AF showed little decrease in the percentage of scratch area over 24 h. However, both uncSFM+10% AF and uncSFM+10% AF exosomes / EVs showed significantly increased closure of the scratch area over 24 h compared to the results of exosome / EV-depleted AF.

[0214] Figure 3 shows Western blot analysis comparing the levels of CD63 and CD9 (markers of exosomes / EVs) in whole amniotic fluid (AF; total), fractions of exosomes purified from AF using the ExoQuick TC-ULTRA kit (SBI Biosciences; exoCrude), purified fractions / eluates from the ExoQuick TC-ULTRA kit (SBI Biosciences; exoPure), and exosome-depleted AF (exo(-)AF). CD63, and to a lesser extent CD9, were present in all fractions except exosome-depleted AF. Albumin is abundant in AF and is therefore used as a marker indicating the removal of contaminants from purified exosomes.

[0215] These experiments show that purified AF-EVs are necessary and sufficient for the wound area closure / migration effects observed in Figures 1 and 2. Migration is important for early stage healing events such as cell recruitment and activation that initiate wound tissue remodeling. Depletion of EVs from AF inhibits migration and epithelial-mesenchymal transition (EMT), as shown by reduced levels of vimentin mRNA in exosome-depleted AF (Figure 4). EV-depleted AF instead promotes re-epithelialization, a "late" event required for wound healing, which serves to return recruited / activated cells to their normal quiescent state.

[0216] These opposing effects are mediated through EMT in AF-EVs but MET in EV-depleted AF. This is supported by the observation that vimentin mRNA (Figure 4; not significantly) and N-cadherin / E-cadherin mRNA ratio (Figure 5; indicating increased EMT) were increased in AF-EV-treated myoblasts but decreased in EV-depleted AF cultures. Evidence for a "mobilization / activation" that can initiate cell / tissue remodeling comes from the observation that levels of Col1a1 (collagen, type I, α1; an abundant collagen present in repaired tissues) (Figure 6) and Acta2 (α-actin-2; a protein involved in cell movement and a marker of myofibroblast formation) (Figure 7) are elevated in AF-EV-treated cells but decreased or unchanged in EV-depleted AF-treated cells. This effect may be mediated by increased TGFβ signaling, as expression of TGFbr2 (transforming growth factor, beta receptor II; a marker of cell proliferation) (Figure 8) is elevated in AF-EV-treated cells but unchanged in EV-depleted AF-treated cells. Conversely, EV-depleted AF showed increased levels of STAT3 (signal transducer and activator of transcription 3; a transcription factor that regulates the expression of genes involved in late wound healing such as epithelial cell proliferation, extracellular matrix remodeling, angiogenesis, and suppression of inflammation), whereas levels in AF-EV-treated cells were unchanged (Figure 9).

[0217] Similar results were observed when the scratch test wound healing assay was repeated using MMM fibroblasts. Figure 10 shows representative brightfield microscopy images at time 0, 12, 18, and 24 hours, and quantification is shown in Figure 11. uncSFM+10% exosome / EV-depleted AF showed little decrease in the percentage of scratch area over 24 hours. However, both uncSFM+10% AF and uncSFM+10% AF exosomes / EVs showed increased closure of the scratch area over 24 hours compared to exosome / EV-depleted AF.

[0218] Conclusions: AF-EVs promote wound healing in an in vitro model by activating EMT. Conversely, EV-depleted AF potently inhibits cell migration and EMT. These findings suggest that a two-step approach to wound treatment, in which AF-EVs are delivered early to induce cell migration and then EV-depleted AF is used to activate later events such as re-epithelialization / MET, may yield superior outcomes.

[0219] Example 2: Topical bioactive cosmetic products preface Current regenerative aesthetic therapies use exfoliation techniques such as microderma-peeling or microneedling to deliver bioactive agents transdermally. Delivery of large biological molecules to the dermis is limited and relies in part on exfoliation and other mechanically disruptive forces. Bioactive agents have historically included autologous platelet-rich plasma (PRP) and growth factor serums, however, they require long (and potentially painful) preparation times, are inconsistent in formulation, and have uncertain safety profiles.

[0220] To circumvent these issues, a treatment was developed and optimized that combines exfoliation and microneedling to disrupt the dermis with a liberal application of an amniotic fluid-based bioactive formulation (commercially available as Celexoderm™ Skin Rejuvenation Serum). Based on the presence of AF components and the body of literature showing that AF can safely promote skin regeneration, it was hypothesized that transdermal delivery of amniotic proteins and other biomolecules via this formulation would reduce wrinkles and tighten the skin.

[0221] The primary objective was to evaluate the wrinkle-smoothing properties of a bioactive hydrogel system (Celexoderm™ Skin Rejuvenating Serum, Merakris Therapeutics, LLC, Research Triangle Park, NC) as an adjunct treatment in subjects undergoing professional facial exfoliation. Secondary objectives were to evaluate skin sensitivity and tolerance to the formulation. method

[0222] Cosmetic formulation: Add the carbomer polymer while stirring the sterile distilled water at approximately 600 rpm with the paddle shaft positioned at an angle of approximately 25° until completely wetted (approximately 45 minutes). Reduce the stirring speed to approximately 300 rpm, then slowly add 1N NaOH until a pH of 6.8-7.0 is reached. Then slowly add Spectrastat™ (1.8% v:v), then slowly add AF to obtain a final concentration of 20% (v:v).

[0223] Subjects (n=3) provided informed consent to undergo professional facial cleansing followed by exfoliation with a 1 mm microneedle roller. Microneedling exfoliates the stratum corneum and creates small microchannels in the dermis, mediating access to the skin's biolayers for topically applied larger biomolecules. Five grams of Celexoderm™ were then applied liberally to the face in a professional setting. Subjects continued to apply Celexoderm™ topically at home by liberally applying the product to targeted wrinkled areas twice daily for 14 days.

[0224] Data was collected in the form of photographs before and 14 days after starting the procedure to assess the overall visual changes in the skin. Subjects also reported qualitative feedback regarding skin sensitivity and product satisfaction to the treating aesthetician. Physician feedback regarding professional application was obtained. result

[0225] Three subjects were treated at a single aesthetic medical center and reported qualitative improvement in skin texture and wrinkle reduction. A photograph of one subject is shown in Figure 12, which shows significant wrinkle reduction and partial disappearance of "crow's feet". Two of the three subjects reported mild burning with Celexoderm™ application after the microneedle exfoliation procedure, but there were no reports of burning during or after application at home (no microneedling). There were no other patient reports of skin sensitivity or other issues. All subjects reported that Celexoderm™ had a desirable feel and texture when applied to the skin. Physicians reported that the product was easy to apply and that the amount of product was sufficient to apply liberally to the face and neck area. Further physician feedback included a suggestion to change the preservative to minimize burning with application, possibly due to the use of an alcohol-based preservative.

[0226] conclusion The results presented herein demonstrate that Celexoderm™ in combination with an exfoliation procedure promotes wrinkle reduction. Due to reports of a burning sensation immediately following treatment with exfoliation (but not reported with home use without exfoliation), the addition of an alcohol-based preservative, USP <61> They were prompted to switch to an alcohol-free preservative that had passed testing (Spectrastat™, Inolex Inc., Philadelphia, PA). Compared to other bioactive formulations such as platelet rich plasma (PRP), utilization of Celexoderm™ reduces safety risks associated with blood draws (zero user discomfort), reduces treatment time in medical spas, and allows for more consistent protein / polymer formulations from batch to batch. Delivery of Celexoderm™ may be enhanced using mechanical forces such as iontophoresis or sonophoresis, which further facilitate penetration of large molecules into the skin.

[0227] We conclude that Celexoderm™ Skin Rejuvenating Serum is a safe and potentially effective off-the-shelf alternative to PRP and other bioactive preparations delivered topically and / or subcutaneously and administered at the point-of-care.

[0228] Example 3.

[0229] Liquid chromatography coupled with tandem mass spectrometry (LC / MS-MS) was performed on triplicate biological samples: total amniotic fluid (total AF), exosome-depleted AF (exo(-)AF), and exosome-enriched fraction of AF (AF exos). Analysis was performed to generate peptide enrichments over the total spectrum and then measure the degree of overlap of peptides that uniquely mapped to proteins. Figure 13 shows the resulting Venn diagram, which indicates the degree of overlap (or non-overlap) of the analyzed samples.

[0230] Using the same "depth" of protein sequencing, the exosomal fraction of amniotic fluid contains a more complex proteome (at the depth of sequencing performed). Proteins highly abundant in AF (e.g., albumin and transferrin) are found primarily outside the exosomal fraction.

[0231] Gene ontology analysis using the DAVID bioinformatics database was used to determine biological values, functions, and processes significantly associated with proteins identified by LC / MS-MS to be present in the exosome-enriched AF fraction at higher levels than proteins found in the total AF. Figure 14 is a plot of log10 (p-value) on the y-axis against frequent terms on the x-axis. The frequent terms identified were, from left to right, cytosol; extracellular exosomes; cell-cell adhesion; involved in cell-cell adhesion; membrane; myelin sheath; GTP binding; GTPase activity; vesicles; actin filament binding, and focal adhesion. Extracellular exosomes (among others) and related terms (e.g., cytosol, membrane, vesicles, actin binding, focal adhesion, cell-cell adhesion, and cadherin binding, etc.) were found. GTP binding / GTPase may be related to several GTP-dependent signaling processes. Gene ontology analysis using the DAVID bioinformatics database was also used to determine biological terms, functions, and processes significantly associated with proteins identified by LC / MS-MS as present in the exosome-depleted AF fraction at higher levels than proteins found in the total AF. Figure 15 is a plot of log10(p-value) on the y-axis against frequent terms on the x-axis. The frequent terms identified are, from left to right, extracellular domain, retinal homeostasis, hormone activity, serine-type endopeptidase inhibitor activity, DNA binding, and positive regulation of blood clotting. There were fewer terms and lower enrichment values ​​than in Figure 14.

[0232] Example 4. Tissue Damage

[0233] Degenerative disorders of the joints, such as osteoarthritis, rheumatoid arthritis, and psoriatic arthritis, result in persistent pain and disability.

[0234] OA is characterized by the destruction of cartilage and loss of extracellular matrix. Articular cartilage is a tensile load-bearing connective tissue that covers the surfaces of joints. Articular cartilage does not contain blood vessels, nervous tissue, or lymphatic vessels. Chondrocytes, which are spatially separated by a large amount of ECM, are responsible for the synthesis and maintenance of ECM. The repair capacity of cartilage declines with age, as manifested by a decrease in chondrocyte numbers. These changes lead to cartilage degeneration and limit its repair capacity. Catabolic and proinflammatory factors produced by the inflamed synovium alter the balance of anabolism and catabolism of the cartilage matrix, causing cartilage destruction. Alterations in cartilage and subchondral bone lead to further synovitis; progressive synovitis worsens clinical symptoms and stimulates further joint degeneration. (Zhang,R.et al.,Am.J.Trans. Res.(2019)11(10): 6275-89).

[0235] Degeneration of cartilage tissue during the progression of OA is caused by chronic inflammation. It is generally accepted that there is a link between inflammatory cytokines and the development of OA. The expression of matrix metalloproteinases (MMPs) and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTS) is increased. It has been suggested that paracrine secretion of exosomes may play a role in the repair of joint tissue. (Mianehsaz, E. et al., Stem Cell Res. & Therapy (2019) 10: 340).

[0236] Preclinical studies have shown that intra-articular injection of MSCs isolated from either adipose tissue or bone marrow can protect articular cartilage from degeneration and delay the onset of OA (ibid. citing ter Huurne, MI et al, Arthritis Rheumatism (2012) 64(11): 3604-13; Toupet, K. et al., PLoS One (2015) 10(1): e0114962; Murphy, JM, et all, Arthritis Rheumatism (2003) 48(12): 3464-74; Desando, G. et al., Arthritis Res. Ther. (2013) 15(1): R22).

[0237] Numerous studies have been conducted to evaluate the various effects of exosomes on various cells involved in joint diseases (ibid. Anderson, HC et al., Lab Investig. (2010) 90(11): 1549; Chang, YH, Wu, KC, et al., Cell Transplant. (2018) 27(3): 349-63; Li, JJ et al., Nanomaterials (Basel) (2019) 9(2): https: / / doi.org / 103390 / nano9020261; Withrow, J. et al., Arthritis Res. Ther. (2016) 18(1): 286). Cell-derived EVs are isolated from synovial fibroblasts (SFs) extracted from inflamed joints of OA and RA patients and used to investigate their role in cellular processes such as inflammation and cartilage degeneration involved in disease progression (ibid. citing Withrow, J. et al., Arthritis Res. Thera. (2016) 18(1): 286; Li, Z. et al., Cell Physiol. Biochem. (2018) 47(5): 2008-17; Maumus, M. et al., Biochimie (2013) 95(12): 2229-34).

[0238] Domenis et al. investigated the immunomodulatory properties of SF-derived exosomes from end-stage OA patients on macrophages differentiated from human peripheral blood mononuclear cells (PBMCs) (ibid., citing (Mediators Inflamm. (2017) 2017:481-498). Treatment of patient cells with exosomes would induce macrophages to produce a series of chemokines and inflammatory cytokines, e.g., CCL8, IL-1β, MMP12, CCL15, MMP7, and CCL20, leading to cartilage degradation and joint inflammation.

[0239] Kolhe et al. conducted a similar experiment, using articular chondrocytes treated with exosomes derived from SFs of OA patients, and showed a significant decrease in cell viability and expression of anabolic genes (COL2A1, ACAN), and an increase in expression of catabolic and inflammatory genes (IL-6, TNF-α) (ibid., citing Kolhe, R. et al., Sci Rep. (2017) 7(1): 2029).

[0240] Kato et al. investigated whether exosomes mediate the interaction between articular chondrocytes and inflammatory synovial fibroblasts (SFB). Exosomes were isolated from untreated similar fragment pairs (SFB) and similar fragment blocks (SFP) treated or untreated with IL-1β, and then added to normal articular chondrocytes. They showed upregulation of MMP-13 and ADAMTS-5 expression and downregulation of ACAN and COL2A1 in articular chondrocytes when treated with exosomes from IL-1β-treated SFB compared to exosomes from untreated SFB. Furthermore, exosomes from IL-1β-treated SFB caused OA-like changes in both in vitro and in vivo models. (Ibid., citing Kato, T. et al, Arthritis Res. Ther. (2014) 16(4): 8163).

[0241] There is increasing interest in the clinical application of MSCs for various diseases, including the potential to treat joint injury and OA (ibid., Toh, WS, et al., Semin. Cell Dev. Biol. (2017) 67: 56-64; Davatchi, F. et al., Intl J. Rheum Dis. (2016) 19(3): 219-25; Lamo-Espinosa, JM et al., J. Transl. med. (2016) 14(1): 246; Vega, A., et al., Transplantation (2015) 99(8): 1681-90; Ham, O. et al., Intl J. Mol. Sci. (2015) 16(7): 14961-78; Qi, Y. and Qi, Y. et al., Mol. Biol. Rep (2012) 39(5): MSCs are typically isolated from synovium (ibid., citing Koizumi, K. et al., Osteoarthr. Cartil. (2016) 24(8): 1413-22), bone marrow (ibid., citing Van Buul, G. et al., Osteoarthr. Cartil. (2012) 20(10): 1186-96) and adipose tissue (ibid., citing Manferdini, C. et al., Arthritis Rheumatism (2013) 65(5): 1271-81). Researchers have evaluated the efficacy of MSCs in restoring damaged tissue function or alleviating disease symptoms of OA or cartilage damage (ibid., citing Mendicino, M. et al, Cell Stem Cell (2014) 14(2): 141-45; Lee, WY-W, Wang, B, J. Orthop. Trans. (2017) 9: 76-88). Despite the functional enhancement (or even regeneration of joint tissue) observed after transplantation of MSCs into diseased joints, their engraftment and subsequent differentiation into the desired cell type rarely occurred (ibid., citing Wyles, CC, et al, Stem Cells Cloning (2015) 8:117).

[0242] Direct cell transplantation approaches have several problems, including poor cell survival after injection, unpredictability of sustained improvements in cell behavior and cell-cell interactions, and problems with maintaining adequate reservoir banks of cells to allow off-the-shelf treatments (Ibid. citing Heldring, N. et al., Hum. Gene Ther. (2015) 26(8): 506-17). Donor suitability is another issue, as MSCs isolated from older or otherwise unhealthy donors have been found to result in poor performance and proliferation (Ibid. citing Siddappa, R. et al., J. Orthop. Res. (2007) 25(8): 1029-41). Furthermore, induction of senescence, loss of proliferation potential, and reduced differentiation capacity (especially beyond 10-20 population doublings) have been attributed to prolonged ex vivo cell expansion of MSCs prior to transplantation (Ibid., citing Siddappa, R. et al., J. Orthop. Res. (2007) 25(8): 1029-41). There are also problems maintaining the cartilage phenotype of differentiated MSCs and preventing them from expanding towards an osteogenic phenotype due to their genetic programming to undergo calcification after chondrogenic induction as part of the normal process of endochondral ossification (Ibid., citing Dickhut, A. et al., J. Cell Physiol. (2009) 219(1): 219-26). Furthermore, MSCs are sensitive to certain environmental response factors that may adversely affect MSC responses in diseased joint environments. For example, human adipose tissue-derived MSCs reportedly switch to a pro-inflammatory secretome upon treatment with TNF, which may then play a role in enhancing the inflammatory response (Id. citing Lee, MJ et al, J. Proeome Res. (2010) 9(4): 1754-62).

[0243] Recent studies have shown that MSC exosomes can promote the repair of cardiac, liver and skin tissues. (Zhang,R.et al.,Am.J.Trans. Res.(2019)11(10): 6275-89). MSC exosomes have also been reported to mediate cartilage repair and regeneration. For example, Zhang et al. first showed the effect of human embryonic MSC exosomes on cartilage repair. Cartilage defects were induced in the tracheal groove of the distal femur of 12 adult rats. After 12 weeks, the exosome-treated defects showed complete cartilage and subchondral bone recovery, as well as other features such as hyaline cartilage with a regular surface, complete adhesion to the adjacent cartilage, and ECM deposition very similar to age-matched controls. (Ibid.). Cosenza et al. found that exosomes derived from allogeneic BMSCs prevented mice from developing OA by protecting cartilage and bone from degradation (Ibid., citing 92). The mechanisms underlying cartilage regeneration by MSC exosomes and other reported therapeutic effects of MSC exosomes remain to be elucidated (Ibid., citing Cosenza, S. et al., Sci. Rep. (2017) 7: 16214).

[0244] The articular ends of the bones of the limbs are covered with hyaline cartilage, which consists of chondrocytes surrounded by an ECM rich in collagen and proteoglycans. Cartilage is bathed in synovial fluid, which is secreted by fibroblast-like synoviocytes (FLS) that surround the joint. Chronic inflammation of the joint associated with RA and mechanical deterioration of articular cartilage associated with OA each appear to involve changes in EVs circulating within the joint space. Murphy, C. et al., Mol. Aspects. Med. (2018) 60: 123-28).

[0245] Various miRNAs appear to mediate several important pathological processes.

[0246] In RA, miR-155 and miR146a are known to be involved in the development of the disease; both of these miRNAs are stimulated by TNF-α and indirectly affect the inflammatory response, with miR-155 increasing inflammation and miR-146a decreasing inflammation. (Ibid. citing Withrow, J. et al., Arthritis Res. Ther. (2016) 18(1): 286).

[0247] In OA, treatment of FLS cells with IL-1β results in the secretion of EVs and increases the levels of miR-500B, miR-4454, miR-720, miR-199b, and miR-3154 (ibid., citing Kato, T. et al., Arthritis Res. Ther. (2014) 16(4): 163). MicroRNAs detected in EVs in synovial fluid of OA patients differ between men and women and are secreted by FLS. In particular, women with OA show a significant downregulation of miR-26a, which is known to target Toll-like receptors such as TLR3 in articular chondrocytes. Estrogen is known to stimulate the production of miR-26a, whereas estrogen inhibitors suppress the expression of miR-23a (ibid., citing Kolhe, R. et al. Sci. Rep. (2017) 7(1): 20-29).

[0248] The healing process of various joint / orthopedic degenerative and physical overuse conditions is complex, but there are several underlying cellular states and pathways on which regeneration converges. These are the reduction of inflammation, resumption of cellular homeostasis, and the recruitment / activation of various immune / progenitor cell types that act in concert to reverse orthopedic pathology. In vitro modeling of such conditions has been widely used in a variety of contexts, including but not limited to various types of arthritis and physical joint injuries (see Blom et al. Arthritis and Rheumatism (2009) 60(2):501-12; Johnson et al., In vitro models for the study of osteoarthritis, The Veterinary Journal 209 (2016) 40-49). For example, the use of cytokine induction via IL-1B or TNF-A is a widely used method to mimic these conditions in mouse models, and in vitro models using fibroblasts, chondrocytes, myoblasts, synoviocytes, or osteoclasts / blasts (ibid.).

[0249] Additionally, assays may be used to test the return of these cells to homeostasis by removing the serum and replacing them with the active ingredient / test agent.

[0250] According to some embodiments, assays that can be used to assay for reversal of such conditions include, but are not limited to, RT-qPCR, immunofluorescence, immunohistochemistry, ELISA, Western blot, or other assays such as those described by Johnson et al. 2016, or other cell-based or immunoassays for measuring the target biomarker(s), including other anti-inflammatory markers encoding proteins such as TIMP proteins, which are natural inhibitors of matrix metalloproteinases (MMPs, a group of peptidases involved in the degradation of the extracellular matrix), e.g., tissue inhibitor of metalloproteinase 1 (TIMP1), tissue inhibitor of metalloproteinase 2 (TIMP2), tissue inhibitor of metalloproteinase 3 (TIMP3), nuclear factor kappa B subunit 1 (NFKB1), transforming growth factor beta receptor 1 (TGFBR1), transforming growth factor beta receptor 2 (TGFBR2), signal transducer and activator of transcription 3 (STAT3), collagen type 1 alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), fibronectin 1 (FN1), ACTA2 (actin alpha 2, smooth muscle), and other collagen / ECM proteins.

[0251] According to some embodiments, in vitro models using fibroblasts, chondrocytes, myoblasts, synoviocytes, or osteoclasts / blasts may serve as surrogates for tissue damage. According to some embodiments, total AF, exo(-)AF, and AF exos may be administered to the in vitro model to treat an inflammatory condition induced by one or more of the above drugs, reverse an inflammatory condition induced by the above drugs, or promote homeostasis in the absence of serum or other anti-inflammatory components (as measured by promoting cell division / proliferation or inhibiting apoptosis in vitro). According to some embodiments, the in vtro model may be maintained under conditions that preclude inflammation or damage.

[0252] Example 5. Reduction of scarring in degenerative eye conditions

[0253] definition The term "angiogenesis" as used herein refers to the growth of new blood vessels from pre-existing vessels. Under physiological conditions, angiogenesis is activated by certain angiogenic molecules such as basic and acidic FGF, VEGF, angiogenin, TGFβ, IFNβ, TNFα, and PDGF. Angiogenesis can also be suppressed by inhibitory molecules such as IFNα, thalidomide, thrombospondin-1, angiostatin, endostatin, the naturally occurring carboxy-terminal form, the non-catalytic domain of MMP-2 (PEX), transfer RNA (tRNA) synthetase, and pigment epithelium-derived factor (PEDF). The normally quiescent capillary system is believed to be tightly controlled by the balance between these naturally occurring stimulators and angiogenesis inhibitors. When this balance is disrupted (e.g., diabetic retinopathy (DR)), capillary endothelial cells are induced to proliferate, migrate, and differentiate.

[0254] The term "fibrosis" is used to describe the fibroblast-mediated wound healing process in non-CNS tissues. It refers to the formation or development of excess fibrous connective tissue as a result of injury or inflammation, or as a result of disruption of its blood supply. It may be the result of a normal healing response that leads to scarring, an abnormal reactive process, or may have no known or understood cause.

[0255] The term "gliosis" as used herein refers to the glial cell-mediated wound healing response observed in the CNS, just as fibrosis is used to describe a similar process in non-CNS tissues. (Friedlander, M., J. Clin. Invest. (2007) 117(3): 576-86). The ocular response to hypoxia and inflammatory insults usually results in retinal or choroidal neovascularization. During development, this process is highly regulated, resulting in the establishment of a well-organized, mature vasculature. In adults, this is often not the case, and associated glial cells (e.g., astrocytes, microglia, Müller glia) proliferate with endothelial cells, causing fibrosis and scar formation. (Ibid.).

[0256] As used herein, the term "retinal gliosis" refers to the proliferation of astrocytes, Muller cells, and / or microglia, which may occur in various retinal layers with focal to diffuse distribution. It is characterized by an increase in the number of retinal glial cells. Retinal gliosis may occur as a primary change (generally of unknown etiology) or as a feature not associated with other retinal pathologies (e.g., degeneration).

[0257] The term "Müller (or Muller) cells" as used herein refers to radial glial cells in the vertebrate retina that have a cylindrical, fibrous shape and span the entire thickness of the retina. Muller cells have a higher refractive index than the surrounding tissue and are oriented along the direction of light propagation, i.e., the path of light through the retina from the vitreous where the light enters the tissue to the external limiting membrane. The inner segments of the photoreceptor cells receive the incoming light. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo has shown that these cells provide low-scattering passage of light from the retinal surface to the photoreceptor cells. Individual Muller cells appear to function as optical fibers, mediating image transfer through the vertebrate retina with minimal distortion and low losses. See Franze, K. et al, "Muller cells are living optical fibers in the vertebrate retina," Proc.Natl Acad.Sci.USA(2017)104(20): 8287-92.

[0258] The term "angiogenesis" as used herein refers to the development of new blood vessels, especially in tissues where circulation is impaired by disease or trauma.For example, corneal neovascularization is characterized by the invasion of new blood vessels into the cornea, and is caused by the disruption of the balance between angiogenic and antiangiogenic factors that maintain the transparency of the cornea.Neovascularization of the iris (NVI), also known as rubeosis iridis, occurs when small, thin blood vessels develop on the anterior surface of the iris in response to retinal ischemia.The term "retinal neovascularization" refers to abnormal blood vessel growth in the retina.

[0259] As used herein, the term "scar tissue" refers to fibrous tissue that replaces normal tissue destroyed by injury or disease as a result of the biological process of wound repair.

[0260] Ocular fibrotic disorders Ocular fibrosis can have devastating consequences for vision, either by mechanically disrupting the visual axis or by sufficiently disrupting the tissue microenvironment that proper cellular function is no longer possible. (Friedlander, M. "Fibrosis and diseases of the eye," J. Clin. Invest. (2007) 117(3): 576-86).

[0261] Anterior segment fibrotic diseases of the eye The response of the anterior segment to wound healing is more similar to the response of non-CNS tissues. The two major diseases of the anterior segment that lead to vision loss are corneal opacities and glaucoma. (Id.)

[0262] The cornea is covered on the outside by a stratified non-keratinizing epithelium and on the inside by a single layer of transporting endothelium sandwiched between multiple orthogonal arrays of collagen. It is normally avascular due to the high concentration of soluble VEGFR-1 and is surrounded by a transitional edge, the limbus, where neoendothelial cells and corneal epithelial stem cells reside. (Ibid.)

[0263] Diseases of the cornea may be hereditary (e.g., hereditary dystrophies) or acquired secondary to infection (e.g., herpetic hepatitis) or inflammation (e.g., pterygium). (Ibid.) Elastic degeneration of the conjunctiva resulting in pinguecula and pterygium (fibrovascular growths on the corneal surface) may result in induced astigmatism and / or loss of vision secondary to occlusion of the visual axis. (Ibid.) The final common event in all these diseases is inflammatory changes, often associated with neovascularization, tissue edema, and ultimately fibrosis of the corneal stroma, which results in opacification and loss of vision. (Ibid.)

[0264] Corneal wound repair is a complex, multiphase process involving apoptosis (Klingeborn, M. et al., Prog. Retin. Eye Res. (2017) 59: 158-77, citing Netto, MV, et al., Cornea (2005) 24: 5009-22); proliferation (ibid., citing Cursiefen, C. et al. Cornea (2006) 25: 443-47); cell transformation (ibid., citing Mimura, T. et al., J. Vasc. Res. (2009) 46: 541-550); migration (ibid., citing Cornea (2006) 25: 443-47); and ECM remodeling (ibid., citing Mimura, T. et al., J. Vasc. Res. (2009) 46: 541-550). A key component of this entire process is the transmembrane matrix metalloproteinase-14 (MMP-14). Corneal fibroblasts release exosomes with MMP-14 that are taken up by endothelial cells (Ibid., citing Han,KY,et al. Invest. Ophthalmol. Vis. Sci(2015)56: 5323-5329). Exosomal MMP-14 activity is important for the accumulation and activation of MMP-2 in exosomes (Ibid., citing Han,KY,et al.Invest. Ophthalmol. Vis. Sci(2015)56: 5323-5329).

[0265] Corneal transplants have countered the uniformly dismal prognosis for patients with clouded or damaged corneas, but they have a high failure rate, usually due to recurrence of the clouding. Id.

[0266] Glaucoma is usually associated with increased intraocular pressure from either increased production of intraocular fluid or increased resistance to outflow, but progressive fibrosis of the canals through which intraocular fluid leaves the eye (trabecular meshwork) is more commonly thought to account for most of the damage that causes glaucoma.

[0267] Posterior segment fibrotic disease of the eye. The posterior segment consists of structures behind the lens; the interior of the rear of the eye is filled with the vitreous, a viscoelastic substance composed primarily of water, collagen, and hyaluronic acid. The vitreous acts as a shock absorber for the retina (the most posterior tissue of the eye) and may provide a scaffold for glial and endothelial cells to migrate from their normal intraretinal locations forward across the retinal surface and / or into the specific vitreous in certain disease states (e.g., diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity (ROP)).

[0268] Diseases that result in vision loss as a result of abnormalities in the retinal or choroidal vasculature (e.g., age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and neovascular glaucoma) are characterized by macular edema, retinal and extracorporeal hemorrhage, and fibrovascular scarring. The final pathophysiological common factor shared by all these diseases is the retinal response to injury, where chronic wound healing leads to fibrosis. When abnormal blood vessels form in response to inflammatory or hypoxic stimuli, they leak fluid, causing retinal thickening and edema and / or hemorrhage, which can lead to fibrovascular proliferation and traction retinal detachment.

[0269] Retinal fibrovascular scarring and gliosis Fibrovascular scars are the result of underlying inflammatory or hypoxic angiogenesis and associated fibrosis. Glial cells are the major participants in the formation of fibrous scars in response to retinal injury or disease. In the retina, specific glia are closely associated with vascular endothelium in both developing and mature tissues. For example, activated astrocytes form a template to which retinal vascular endothelial cells migrate during the formation of the superficial vascular plexus; any disturbance in the number or distribution of these cells disrupts the normal development of the retinal vasculature. Inflammatory diseases (e.g., AMD and ischemic diseases (e.g., DR)) account for most of the pathologies that lead to retinal fibrovascular scars and associated vision loss.

[0270] Subretinal fibrosis: AMD When the retinal pigment epithelium (RPE) ages or becomes diseased, it can function improperly, leading to the accumulation of subretinal deposits called drusen, which contain neovascular lipids and damaged proteins. RPE dysfunction and drusen accumulation can lead to thickening of Bruch's membrane, a shiny, homogenous membrane between the outer portion of the retina (the choriocapillaris) and a layer of capillaries lined by fenestrated type II endothelium that nourishes the retina, and the accumulation of neovascular drusen associated with this fibrosis can reduce oxygen diffusion from the choriocapillaries to the photoreceptors, further exacerbating the condition and resulting in choroidal neovascularization. As these abnormal new blood vessels begin to grow in the subretinal space, they often bleed, triggering further wound-healing responses and ultimately causing subretinal fibrosis. Localized destruction of the photoreceptors, RPE, and choroidal vessels results in permanent impairment of macular function and vision. Although rodents do not appear to faithfully mimic human disease, transgenic mice offer some utility (Pennesi, ME, et al., Mol. Aspects Med. (212) 33 (40: 487-509).

[0271] Current therapies for treating AMD-associated choroidal neovascularization and DR aim to inhibit cytokines that mediate the vascular proliferative response or destroy the tissues causing increased metabolic demand, but inhibition of angiogenic cytokines does not address the ischemic and inflammatory stimuli that underlie the pathophysiology.

[0272] Epiretinal fibrosis: DR In DR, ischemia develops as a result of diabetic microvascular disorders, such as pericyte death, microaneurysms, intraretinal microvascular abnormalities, altered vascular permeability, and macular edema. Increased hypoxia can result in neovascularization, leading to intraretinal, subvitreal (between the retinal surface and the posterior vitreous base), and vitreous hemorrhage. These proliferating blood vessels are accompanied by gliosis. As abnormal blood vessels continue to proliferate at the retinal surface, they stretch and contract in the vitreous, causing traction on the retinal surface, which can lead to retinal detachment.

[0273] Although animal models of ischemic retinopathy have helped to develop a better understanding of the factors that control retinal vascular growth, no model fully recapitulates the complete pathology of the neuronal and vascular changes that occur at each stage of diabetic retinopathy. (Olivares, AM, et al., Curr. Diab. Rep. (2017) 17 (10): 93)

[0274] Efforts to minimize sub- and epiretinal fibrosis have met with limited success, and therapeutic intervention comes too late to save vision, as scarring has already led to photoreceptor death.

[0275] Retinal neovascularization and associated gliosis and fibrosis are also observed in ROP and as a complication of surgery to treat retinal detachment.

[0276] 9. Retinal Damage, Detachment, and PVR Ocular trauma is the second most common cause of vision impairment in the United States, with approximately 2.4 million injuries occurring annually, 10-20% of which result in temporary or permanent vision loss (US EyeInjury Registry. 2016;American Academy of Ophthalmology. 2016). Many forms of traumatic injury to the eye, such as penetrating trauma and percussive head injuries, cause tears in the retina and subsequent detachment from its primary source of metabolic support, the retinal pigment epithelium (RPE) and choroidal vasculature. Retinal detachment inevitably results in degeneration of photoreceptor cells and some loss of vision. Small detachments may resolve spontaneously, but minimization of vision loss is best ensured by timely surgical repair. In addition to the loss of vision associated with the initial injury, 15.7% of retinal damage may result in a secondary blinding condition caused by the growth of scar tissue, consisting mainly of RPE and glial cells, at the subretinal and / or epiretinal surfaces of the retina (Miura, M. et al. Retina (2000) 20(5): 456-58). Scar tissue formation at either surface is considered part of the spectrum of neoplastic fibrocontractile retinopathies called proliferative vitreoretinopathy or PVR (Machemer, R. et al., Arch. Ophthalmol. (1991) 109(11): 1492-93). Subretinal scars disrupt retinal function and vision by acting as a barrier between the RPE and retina after reattachment surgery, thus preventing phagocytosis of photoreceptor outer segments, completion of the retinoid cycle, and retina-RPE-choroid transport. The epiretinal membrane obscures light and may shrink, causing retinal folds and retinal redetachment.Epiretinal membrane formation and subsequent shrinkage detachment remain the most common failure of retinal reattachment surgery (Speicher,MA et al. Retina(2000)20(5): 459-64;Duquesne,N.et al.,Graefes Arch. Clin.Exp. Ophthalmol.(1996)234(11): 677-82;Girard,P.,et al.,Retina(1994)14(5): 417-24;Gartry,DS,et al,Br. J.Ophthalmol(1993)77(4): 199-203;Greven,CM et al,Ophthalmology(1992)99(2): 257-62). Advances in surgical management have improved the ability to ultimately reattach the retina after the occurrence of shrinkage detachment, but the visual prognosis remains poor.

[0277] Early cellular events in retinal detachment and PVR Although PVR-induced scarring of the retina is associated with the greatest loss of vision after retinal detachment, significant and often irreversible changes in the retina and RPE begin immediately after injury. Within hours of detachment, there is increased Müller cell expression of transcription factors involved in both proliferation and cell growth (Geller, SF et al., Invest. Ophthalmol. Vis. Sci. (2001) 42 (6): 1363-69). This is followed by a burst of glial cell proliferation during the first 3 days after injury, followed by scar formation (Lewis, GP et al., Mol. Vis. (2010) 16: 1361-72; Fisher, SK et al., Invest. Ophthalmol. Vis. Sci. (1991) 32 (6): 1739-48). Similarly, proliferation of the RPE has been observed 24 hours after injury, even in cases of experimentally induced detachment, where physical trauma is minimal (Anderson,DH et al.,Invest. Ophthalmol. Vis. Sci.(1981)21(1 Pt1):10-16). Concomitant with these proliferation events is photoreceptor apoptosis and, in the absence of reattachment, eventual remodeling of neurons throughout the injured area (Cook,B.et al.,Invest. Ohthalmol. Vis. Sci.(1995)36(6):990-96;Fisher,SK et al.,In:Kolb H,Fernandez E,Nelson R,(eds)Webvision:The Organization of the Retina and Visual System [Internet].Salt Lake City (UT):University of Utah Health Sciences Center;1995-.). Although retinal reattachment effectively prevents Müller cells from growing into the subretinal space, Müller cell proliferation continues at a low level and can redirect growth to the epiretinal surface (Lewis, GP et al., MOl. Neurobiol. (2003) 28(2): 159-75. The low level of proliferation observed in the surgically reattached retina may explain why PVR scar formation in the vitreous is not usually observed in patients until several months after reattachment surgery.Blocking early cellular events that can adversely affect the retina is therefore essential to maximizing visual recovery after injury.

[0278] Early molecular signals Within minutes of detachment, there is evidence of fibroblast growth factor (FGF)-mediated activation of the mitogen-activated protein kinase (MAPK) pathway in Müller cells and RPE (Geller, SF et al., Invest. Ophthalmol. Vis. Sci. (2001) 42 (6): 1363-69). By the first day, upregulation of phosphorylated signal transducer and activator of transcription 3 (STAT3) is seen in some inner nuclear layer and ganglion cells, and by the third day, it is also detected in some RPE cells. Stretching cultured Müller cells induces expression of genes associated with interleukin 6 (IL6) proliferation and stimulates phosphorylation of STAT3 via the IL6 receptor and Janus kinase (JAK) (Wang, X. et al. PLoS One (2013), 8 (5) e63467), suggesting that deformation of the retina during injury may be the trigger that initiates many of the events associated with injury. Similarly, physical disruption of the epithelial monolayer may be involved in activating RPE proliferation. FGF promotes mitosis in subconfluent RPE in culture, but in fact differentiation in intact RPE monolayers, and differentiated RPE cells exposed to FGF do not enter the cell cycle (Radeke, MJ et al., Genome Med. (2015) 7(1): 58). Normal RPE cells possess tight junctions that sequester cyclin-dependent kinase 4 (CDK4) and Y-box binding protein 3 (YBX3), thus preventing them from entering the cell cycle. Trauma-induced disruption of these tight junctions leads to the release and nuclear relocalization of CDK4 and YBX3, and the G1 / S phase transition.

[0279] Therapies for PVR Scarring PVR is a complex disease involving multiple cell types, unwanted cell proliferation, cell spreading, and contractility. Cell division has been observed in membranes removed from patients with PVR (Tsanou, E. et al., Intl J. Clin. Pract. (2005) 59 (10): 1157-61; Zhang, X et al. Curr. Eye Res. (2005) 30 (5): 395-403; Lesnik Oberstein, SY et al., Br. J. Ophthalmol. (2011) 95 (2): 266-72). Data from animal models, as well as membranes removed from human patients, indicate a critical role for Müller cell proliferation as part of the response. As the growth of Müller cell processes into the retinal surface is routinely observed as one of the earliest events following detachment, these processes may provide a cellular scaffold on which more complex cell membranes can form.

[0280] Studies of retinal detachment in animal models suggest a sequence of events that ultimately leads to PVR: retinal detachment causes 1) intraretinal proliferation and hypertrophy of Müller cells, 2) "proliferation" of Müller cells into the subretinal space, formation of an extensive glial scar, 3) eventual migration of retinal pigment epithelial cells to the membrane, and 4) integration of immune cells into the membrane and the retina (Fisher, SK et al., In: Kolb H, Fernandez E, Nelson R (eds.) Webvision: The Organization of the Retina and Visual System [Internet]. Salt Lake City (UT): University of Utah Health Sciences Center; 1995-2005; Fisher, SK, Lewis, GP, Vision Res. (2003) 43(8): 887-97).

[0281] The pathophysiological fibrotic response in retinal detachment is largely mediated by RPE cells following exposure to numerous growth factors and cytokines found in the vitreous (Sadaka A & Giuliari G.,Cllin. Ophthalmol.(2012)6: 1325-33;Moysidis S,et al. Mediators Inflamm.(2012)2012.815937). These factors promote an environment for cell migration, proliferation, survival and the formation of extracellular proteins.

[0282] The role of extracellular vesicles in vivo Infiltrating and / or localized monocytes have been implicated in a wide range of ocular diseases, such as choroidal neovascularization ((Klingeborn, M. et al., Prog. Retin. Eye Res. (2017) 59: 158-77, citing Espinosa-Heidmann, DG, et al. Invest. Ophthalmol. Vis. Sci. (2003) 44: 3586-92); uveitis (Lee, RW., et al., Semin. Immunopathol. (2014) 36: 581-94); keratitis (ibid., Cursiefen, C., et al., J. Clin. Invest. (2004) 113: 1040-50; Cursiefen, C., et al., J. Exp. Med. (2011) 208: 1083-92; Koch, A. E., et al., Science (1992)258: 1798-1801); diabetic retinopathy (ibid., McLeod, DS., et al., Am. J. Pathol. (1995) 147: 642-53; Schroder, S., et al Am. J. Pathol. (1991) 139: 81-100; Serra, AM., et al. al., Am. J. Pathol. (2012) 181:719-27), and glaucoma (ibid., Alvarado, J. A., et al., Arch. Ophthalmol. (2010) 128: 731-37; Howell, GR., et al., J. Clin. Invest. (2012) 122: Cited 1246-61. It has been suggested that under homeostatic conditions, RPE-derived extracellular vesicles (EVs) can downregulate immune activity in the immediate vicinity of retinal pigment epithelial cells. (Klingeborn, M. et al., Prog. Retin. Eye Res. (2017) 59: 158-77).

[0283] Few studies have been conducted to investigate the role of exosomes in the development and disease process of AMD, including choroidal neovascularization (CNV), or other diseases with abnormal retinal neovascularization such as DR. (Klingeborn, M., et al., Progr. Eye Res. (2017) 59: 158-77). A delicate balance of pro- and anti-angiogenic signaling exists in the retina, RPE, and choroid. (Ibid.). The role of exosomes in this signaling balance was highlighted by a study showing that exosomes released from retinal astrocytes contain anti-angiogenic components that inhibit laser-induced CNV in a mouse model (Ibid., citing Hajrasouliha, AR., et al J. Biol. Chem. (2013) 288: 28058-067).

[0284] Aqueous humor (AH) has been used for protein, nucleic acid, and lipid biomarker analysis in ocular diseases such as glaucoma (ibid., Agnifili, L. et al., Progr. Brain Res. (2015) 221:1-32; Goyal, A., et al., Current Eye Res. (2014) 39: 823-29); neovascular AMD (ibid., Kang, GY et al., J. Proteome Res. (2014) 13:581-95; Liu, F. et al., Mol. Vis. (2016) 22: 352-61; Park, KH, et al., Invest, Ophthalmol. Vis. Sci. (2014) 55: 5522-30 (2014); diabetes-induced eye disease (ibid., Vijosevic, S. et al., Invest. Ophthalmol. Vis. Sci.(2015)56: 1913-18; citing Vijosevic,S.et al.,Acta Ophthalmol.(2016)94:56-64, and uveitis (ibid., citing Haasnoot,AM et al.,Arthritis Rheumatol.(2016)68: 1769-79; Kalinina Ayuso,V.et al.,Invest. Ophthalmol. Vis. Sci.(2013) 54: 3709-20), most of the nucleic acid and lipid biomarkers, and some of the protein biomarkers identified in AH, were likely exosome-associated, but little attention has been paid to exosome-specific biomarkers due to uncertainties regarding the authenticity of exosome preparations. Ibid.

[0285] MSC and exosome therapy in animal models It was reported that MSCs transplanted into the vitreous after optic nerve crush injury promoted significant neuroprotection of retinal ganglion cells and modest regeneration of their axons. (Mead, B., Tomarev, S. Stem Cell Translational Med. (2017) 6: 1273-85), Levkovitch-Verbin, H., et al. (cited Invest. Ophthalmol. Vis. Sci. (2010 51: 6394-6400; Mead, B., et al., Invest. Ophthalmol. Vis. Sci. (2013) 54: 7544-56; Tan, HB, et al., Clin. Interv. Aging (2015) 10: 487-90; Zwart, I., et al., Exp. Neurol. (2009) 216: 439-448). MSCs have also been reported to promote the survival of retinal ganglion cells and their axons and maintain their function in animal models of glaucoma. (Ibid., Mead, B. et al. al,Cytotherapy(2016)18: 487-96;Emre,E.et al.,Cytotherapy(2015)17: 543-59;Johnson,TV,et al.,Invest. Ophthalmol. Vis. Sci.(2010)51: 2051-59;Yu,S.et al.,Biochem. Biophys. Res.Commun.(2006)344: 1071-79). Such studies strongly suggest paracrine mechanisms rather than cell replacement as the primary mechanism of such effects. Accumulating data support the idea that MSC-derived exosomes can mediate the biological functions of MSCs.

[0286] For example, exosomes derived from bone marrow-derived MSCs cultured under hypoxic conditions containing angiogenic proteins and growth factors were used to determine the effect of intravitreal administration on retinal ischemia in a mouse model. Oxygen-induced retinopathy was induced in C57BL / 6J mice. The right eye of each mouse was intravitreally injected with 1 μl of saline or exosomes derived from human MSCs and compared with control mice. Two weeks after injection, retinal perfusion was evaluated. Intravitreal exosome treatment partially preserved retinal vascular flow in vivo and reduced associated retinal thinning. Retinal neovascularization was reduced when compared with saline-treated eyes. No immunogenic or ocular / systemic side effects were associated with this treatment. Moisseiev, E. et al., Current Eye Res. (2017) 42 (10): 1358-67).

[0287] Exosomes derived from pooled human bone marrow-derived MSCs were tested by another group in an in vitro model of retinal ganglion cell (RGC) death and axon regeneration, and in a rat optic nerve crush model, to test their neuroprotective and axonogenic potential, and whether the effects were protein- or miRNA-mediated. (Mead, B., Tomarev, S., Stem Cell Translational Med. (2017) 6: 1273-85). Significant neuroprotective and neuritogenic effects were reported by treating primary retinal cultures. Twenty-one days after optic nerve crush and weekly intravitreal exosome injections, optical coherence tomography, electroretinograms, and immunohistochemistry showed that exosomes promoted statistically significant survival of RGCs and regeneration of their axons, while RGC axon loss and RGC dysfunction were partially prevented. The effects were dependent on miRNA-dependent mechanisms, as shown by the reduced therapeutic effect after knockdown of Aragonaute-2, a key miRNA effector model. (Ibid.).

[0288] A rat retinal detachment (RD) model developed using subretinal injection of 1% hyaluronic acid in male Sprague-Dawley rats has also been used to investigate the therapeutic effects of exosomes derived from rat bone marrow MSCs. (Ma, M. et al., Exp. Eye Res. (2020) 191: 107899). For treatment, various concentrations of MSC-exosomes (5 μL) were injected into the subretinal space immediately after surgical retina-RPE separation and compared with 5 μL of PBS control. Three days after retinal detachment and MSC-exosome treatment, retinas were dissected, immediately frozen in liquid nitrogen, and total RNA was extracted using Trizol (Invitrogen, USA). cDNA was synthesized using the Revertaid kit for first strand cDNA synthesis (Thermo, USA), and qRT-PCR was performed. Retinal expression levels of inflammatory cytokines TNF-α, IL-1β, and monocyte chemotactic protein-1 (MCP-1) were detected by RT-PCR, autophagy-related protein 5 (Atg5) and microtubule-associated protein 1 light chain 3β (LC3) were detected by Western blot, and apoptosis was examined using a TUNEL assay 3 days after RD. Retinal structures were observed 7 days after RD. Proteomic analysis was also performed to detect proteins carried by MSC-derived exosomes using iTRAQ-based technology combined with one-dimensional nano-LC-nano-ESI-MS / MS. It was found that the expression of TNF-α and IL-1β was significantly decreased after MSC-derived exosome treatment. The ratio of LC3-II (active form) to LC3-I (inactive form), which indicates autophagy (representing a protective effect against cell damage), was enhanced, and the cleavage of Atg5 was decreased. Treatment with MSC-derived exosomes also inhibited photoreceptor cell apoptosis and maintained normal retinal structure when compared with the control group.Proteomic analysis revealed that MSC-derived exosomes contained 683 candidate proteins from three biological replicates, which were clustered into 43 biological processes, including cell adhesion, immune response, cytoskeleton remodeling, and development, as well as cell proliferation and differentiation, which may contribute to the therapeutic effect of MSC-derived exosomes in improving photoreceptor cell degeneration.Nine of the 193 proteins exhibited anti-inflammatory, neuroprotective, and anti-apoptotic effects and were hypothesized to play an important role in the therapeutic efficacy of retinal detachment.

[0289] In summary, a variety of specific and generalized pathologies can result in ocular dysfunction. Many in vitro models exist in which ocular dysfunction and its reversal can be tested. Measurements include reversal of inflammation, prevention of apoptosis, promotion of cell proliferation, or other types of relevant readouts. In vitro models using corneal fibroblasts (see, for example, Karamichos et al Invest Opthalamol Vis Sci (2010) 51 (1382-88) and keratocytes (see, for example, Chawla and Ghosh J Cell Phys (2018) 233; 3817-30) are available in which degenerative, fibrotic, and irritative conditions can be tested. Additionally, age-related macular degeneration (AMD) pathology can be modeled using pluripotent or embryo-derived retinal pigment epithelial cells (Forrest et al Dis Models Mech (2015) 8, 421-7).

[0290] One commonality between these disease states / models is that measurements of cell viability / proliferation can be used as a surrogate for cell homeostasis in the absence of serum / sustaining media reagents by replacing AF, AFexos, or exo(-)AF. If cell viability / proliferation is maintained, it is concluded that the test reagent may be effective in maintaining cell homeostasis and reversing the degeneration, fibrosis, irritation, and / or other pathologies that lead to AMD.

[0291] According to some embodiments, exemplary test reagents for inducing degeneration, fibrosis, stimulation, etc. can be stimulatory or pro-inflammatory agents such as, but not limited to, IL-1, TNF-α, vitamin C, TGFB1, IL-6, or IL-8.

[0292] Reversal of pathology, including degeneration, fibrosis, or irritation, can be measured following incubation of an in vitro cell model with a test agent as described above, and cell viability or apoptosis can be measured. Measurements at the molecular or cellular level include RT-qPCR, ELISA, immunofluorescence, immunohistochemistry, western blot, or other assays to measure the reduction of markers and / or changes in other downstream biomarkers such as TIMP1, TIMP2, TIMP3, NFKB1, TGBFR1, TGBFR2, STAT3, COL1A1, COL1A2, FN1, ACTA2, other collagens / ECM proteins various collagens, smooth muscle actin, TGF-β, SMADs (a group of related intracellular proteins important in transduction of transforming growth factor β (TGFβ) superfamily information at the cell surface to the nucleus signal; see, e.g., Attisano, L., Lee-Hoeflich, ST, Genome Biol. (2001) 2(8): PMC138956), fibronectin, and E-cad / N-cad.

[0293] According to some embodiments, a change in one or more biomarkers in the presence of the test agent(s) may indicate that the test agent may reverse the modeled ocular pathological condition.

[0294] Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A method for promoting wound healing in a subject in need thereof, comprising contacting wounded tissue of the subject with a first composition comprising a therapeutic amount of extracellular vesicles (EVs) derived from human amniotic fluid (AF), wherein the therapeutic amount is effective to reduce wound area and promote repair of the wound tissue.

2. 10. The method of claim 1, wherein the composition is effective to promote wound healing by activating epithelial cells to transform into a mesenchymal cell phenotype (EMT).

3. The method of claim 1, wherein the EVs are derived from amniotic fluid mesenchymal stem cells (MSCs).

4. The EV is 10. The method of claim 1, characterized by a sedimentation at about 100,000 x g, a buoyant density in sucrose of about 1.10 to 1.21 g / ml, and an average diameter of about 50 nm to about 200 nm.

5. The method of claim 1 , wherein the contacting is topical or subcutaneous.

6. The method of claim 1, wherein the first composition is effective to increase mRNA levels of one or more of vimentin, N-cadherin, Col1a1, Acta2, or TGFbr2.

7. 13. The method of claim 1, further comprising contacting the wound tissue of the subject with a second composition comprising a therapeutic amount of EV-depleted AF, wherein the therapeutic amount of the second composition is effective to activate mesenchymal-epithelial transition (MET) and promote repair of the wound tissue.

8. 8. The method of claim 7, wherein the length of time between contacting the tissue with the first composition and the second composition is from about 4 hours to about 24 hours.

9. 8. The method of claim 7, wherein the second composition is effective to increase Stat3 mRNA levels.

10. The method of claim 1 , wherein the wound is a chronic wound.

11. The method of claim 10, wherein the wound is a diabetic ulcer, a pressure ulcer, or a venous ulcer.

12. The method of claim 1 , wherein the wound is a burn.

13. The method of claim 1 , wherein the composition further comprises a pharma- ceutically acceptable carrier.

14. 2. The method of claim 1, wherein the EVs are purified from the amniotic fluid by one or more of: a) ultracentrifugation; b) sucrose density gradient centrifugation; c) column chromatography; d) size exclusion; or e) filtration through a device comprising an affinity matrix selective for EVs.

15. The method of claim 14 , further comprising filtering the EVs by size.

16. The method of claim 15, wherein the EVs are characterized by an average diameter of about 50 nm to about 200 nm.

17. 1. A two-step method for promoting wound healing in a subject in need thereof, comprising, in order: a. contacting the wound with a composition comprising amniotic fluid (AF)-derived extracellular vesicles (EVs) to promote early stage wound healing in the subject; b) the two-step method comprising contacting the wound with a composition comprising EV-depleted AF to promote late stage wound healing in the subject.

18. 18. The two-stage method of claim 17, wherein an early stage of wound healing is characterized by activating epithelial-mesenchymal transition (EMT) and inducing cell migration, and a later stage of wound healing is characterized by activating mesenchymal-epithelial transition (MET) and epithelialization of the wound.

19. The method of claim 17, wherein the EVs are derived from amniotic fluid mesenchymal stem cells (MSCs).

20. The EV is 18. The method of claim 17, characterized by a sedimentation at about 100,000 x g, a buoyant density in sucrose of about 1.10 to 1.21 g / ml, and an average diameter of about 50 nm to about 200 nm.

21. 18. The method of claim 17, wherein the contacting is topical or subcutaneous.

22. A method for regulating skin condition in a subject in need thereof, comprising contacting the skin of the subject with a composition comprising a therapeutic amount of extracellular vesicles (EVs) derived from human amniotic fluid (AF), wherein the therapeutic amount is effective to improve skin texture, reduce wrinkles, or both, thereby regulating the skin condition.

23. 23. The method of claim 22, further comprising microneedling the skin prior to contacting with the composition.

24. 23. The method of claim 22, wherein the composition is effective to regulate the skin condition by activating epithelial-mesenchymal transition (EMT).

Images